N-alkoxyamide conjugates as imaging agents

ABSTRACT

The present disclosure is directed to compounds, diagnostic agents, and related methods. In some cases, methods for treating patients are provided. More specifically, the disclosure provides compounds, diagnostic agents, and kits for detecting and/or imaging and/or monitoring elastin rich tissues. In addition, the disclosure provides methods of detecting and/or imaging and/or monitoring the presence of coronary plaque, carotid plaque, iliac/femoral plaque, aortic plaque, renal artery plaque, plaque of any arterial vessel, aneurism, vasculitis, other diseases of the arterial wall, and/or damage or structural changes in ligaments, uterus, lungs or skin, as indicated by changes in total vessel wall area, internal lumen size, and exterior arterial perimeter.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to co-pendingU.S. Provisional Application Ser. No. 61/019,627, filed Jan. 8, 2008,the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds and diagnostic agents, andrelated methods.

BACKGROUND OF THE INVENTION

Cardiovascular diseases are the leading cause of death in the UnitedStates, accounting annually for more than one million deaths.Atherosclerosis is the major contributor to coronary heart disease and aprimary cause of non-accidental death in Western countries. Considerableeffort has been made in defining the etiology and potential treatment ofatherosclerosis and its consequences, including myocardial infarction,angina, organ failure, and stroke. Despite this effort, there are manyunanswered questions including how and when atherosclerotic lesionsbecome vulnerable and life-threatening, the best point of intervention,and how to detect and monitor the progression of lesions.

In the last two decades, many radiotracers have been developed based onseveral molecules and cell types involved in atherosclerosis. Ingeneral, radiolabeled proteins and platelets have shown some clinicalpotential as imaging agents of atherosclerosis, but due to poortarget/background and target/blood ratios, these agents are not idealfor imaging coronary or even carotid lesions. Radiolabeled peptides,antibody fragments, and metabolic tracers like FDG appear to offer newopportunities for nuclear scintigraphic techniques in the non-invasiveimaging of atherothrombosis. However, a non-invasive method to diagnoseand monitor various cardiovascular diseases is needed.

SUMMARY OF THE INVENTION

The present invention relates to compounds of Formula (I),

or a pharmaceutically acceptable salt thereof, wherein:

X is a heteroatom;

R¹ is hydrogen, alkyl, alkenyl, arylalkyl, alkylarylalkyl, alkoxyalkyl,heteroalkyl, or heterocyclylalkyl;

R² and R³ can be the same or different and are hydrogen, alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; and

R⁴ is alkyl, alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl,arylalkyl, alkylarylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonyl,heteroalkyl, heterocyclyl, or heterocyclylalkyl,

wherein each R¹, R², R³, and R⁴ is unsubstituted or substituted with oneor more of the following: alkyl, alkenyl, cycloalkyl, alkylaryl,alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy, alkoxyalkyl,alkoxycarbonyl, heteroalkyl, heterocyclyl, or heterocyclylalkyl,—NR¹⁹R²⁰, —SH, —S(Pg), —OH, —PR¹⁹R²⁰, —P(O)R²¹R²², —CO₂H, ═O, halo,trifluoromethyl, cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO,—CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴,—NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H,—SO₂R²⁴, —SR²⁴, —S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴,NO₂, —C(═O)NHOR²⁴, —C(═O)NHNR²⁴R²⁴, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,or a chelator moiety;

R¹⁹ and R²⁰ are each independently selected from hydrogen, C₁₋₁₀alkylsubstituted with 0-3 R²³, aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkylsubstituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkyl substituted with 0-3R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³, and heterocyclylsubstituted with 0-3 R²³.

R²¹ and R²² are each independently selected from —OH, C₁₋₁₀alkylsubstituted with 0-3 R²³, aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkylsubstituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkyl substituted with 0-3R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³, and heterocyclylsubstituted with 0-3 R²³,

each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHNR²⁴R²⁴, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl;

each R²⁴ is independently selected from hydrogen, alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl;

Pg is a thiol protecting group; and

n′ is an integer from 0-4,

wherein the compound comprises at least one chelator moiety.

In some embodiments, X is nitrogen. In some embodiments, X is oxygen. Insome embodiments, X is sulfur. In some embodiments, X is phosphorus.

In some embodiments, n′ is an integer from 0-3.

In some embodiments, each R²⁴ is independently hydrogen, C₁₋₆alkyl,phenyl, benzyl, or C₁₋₆ alkoxy.

In one set of embodiments,

X is nitrogen;

R¹ is hydrogen, alkyl, arylalkyl, or alkylarylalkyl;

R² and R³ can be the same or different and are hydrogen, alkyl,alkylaryl, aryl, arylalkyl, alkylarylalkyl, or heterocyclylalkyl;

R⁴ is alkyl, alkylaryl, aryl, arylalkyl, or alkylarylalkyl,

wherein at least one of R¹, R², R³, and R⁴ is substituted with achelator moiety;

In any of the foregoing embodiments, R² or R³ can comprise the followingstructure,

wherein

n is 0-6; and

R^(z) is selected from alkyl, aryl, cycloalkenyl, cycloalkyl,heteroaryl, and heterocyclyl.

In any of the foregoing embodiments, R² or R³ can also comprise thefollowing structure,

wherein

n is 0-6;

R^(y) is selected from hydrogen, alkenyl, and alkyl; and

R^(z) is selected from alkyl, aryl, cycloalkenyl, cycloalkyl,heteroaryl, and heterocyclyl.

In one set of embodiments,

n is 1 or 2;

R^(y) is hydrogen; and

R^(z) is selected from alkyl, aryl, cycloalkyl, and heteroaryl.

In some embodiments, R¹ comprises the at least one chelator moiety. Insome embodiments, R² or R³ comprises the at least one chelator moiety.In some embodiments, R⁴ comprises the at least one chelator moiety.

In one set of embodiments, the compound has a structure as in Formula(II),

or a pharmaceutically acceptable salt thereof; wherein

R⁴ is alkyl, alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl,arylalkyl, alkylarylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonyl,heteroalkyl, heterocyclyl, or heterocyclylalkyl, substituted with the atleast one chelator moiety;

n is 0-6;

R^(y) is selected from hydrogen, alkenyl, and alkyl; and

R^(z) is selected from alkyl, aryl, cycloalkenyl, cycloalkyl,heteroaryl, and heterocyclyl.

In another set of embodiments, the compound has a structure as inFormula (III),

or a pharmaceutically acceptable salt thereof, wherein

R² and R³ can be the same or different and are hydrogen, alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl, and at least one of R² and R³ issubstituted with the at least one chelator moiety; and

R⁴ is alkyl or arylalkyl.

In another set of embodiments, the compound has a structure as inFormula (IV),

or a pharmaceutically acceptable salt thereof; wherein

R¹ is alkyl, alkenyl, cycloalkyl, arylalkyl, alkoxyalkyl, heteroalkyl,or heterocyclylalkyl, substituted with the at least one chelator moiety;

n is 0-6;

R^(z) is selected from alkyl, aryl, cycloalkenyl, cycloalkyl,heteroaryl, and heterocyclyl; and

R⁴ is alkyl or arylalkyl.

In any of the foregoing embodiments, the at least one chelator moietyhas the structure,

wherein X′ is a heteroatom; and D¹ and D² can be the same or differentand are hydrogen or a chelator moiety. In some embodiments, the at leastone chelator moiety has the structure,

wherein D¹ and D² can be the same or different and are hydrogen or achelator moiety. In some embodiments, one of D¹ and D² is a hydrogen andthe other is a chelator moiety. The chelator moiety may be selected from

wherein o, p, q, r, s, t, and u are each independently 1-6; and v, w, x,and y are each independently 1-3. In some embodiments, o, r, s, t, and uare each 1; and p and q are each 2. In some embodiments, o, r, s, t, v,w, x and y are each 1.

In one embodiment, the compound has the structure,

In another embodiment, the compound has the structure,

In another embodiment, the compound has the structure,

In another embodiment, the compound has the structure,

In another embodiment, the compound has the structure,

In another embodiment, the compound has the structure,

In one aspect of the present disclosure is provided a compound ofFormula (I-A),

or a pharmaceutically acceptable salt thereof; wherein

A is a D-amino acid residue or a peptide consisting of a D-amino acidresidue and a second D-amino acid;

D¹ and D² are independently selected from hydrogen, a chelator moiety,and an imaging moiety; and

L¹ is a linker; or

L¹ and D¹, together with the nitrogen atom to which they are attached,form a five- to seven-membered ring.

In a first embodiment of the first aspect L¹ is a linker selected fromalkenylene, alkylarylalkylene, alkylene, arylalkylene, heteroalkylene,and heterocyclylene. In a second embodiment of the first aspect L¹ isalkylene. In a third embodiment of the first aspect L¹ is arylalkylene.In a fourth embodiment of the first aspect L¹ is alkylarylalkylene.

In a fifth embodiment of the first aspect A is a D-amino acid residue.In a sixth embodiment of the first aspect A is

wherein

n is 0-6;

R^(y) is selected from hydrogen, alkenyl, and alkyl; and

R^(z) is selected from alkyl, aryl, cycloalkenyl, cycloalkyl,heteroaryl, and heterocyclyl. In a seventh embodiment of the firstaspect n is 1 or 2; R^(y) is hydrogen; and R^(z) is selected from alkyl,aryl, cycloalkyl, and heteroaryl.

In an eighth embodiment of the first aspect the present disclosureprovides a compound wherein one of D¹ and D² is a hydrogen and the otheris a chelator moiety. In a ninth embodiment of the first aspect one ofD¹ and D² is hydrogen and the other is a chelator moiety selected from

wherein

o, p, q, r, s, t, and u are each independently 1-6; and

v, w, x, and y are each independently 1-3.

In a tenth embodiment o, r, s, t, and u are each 1; and p and q are each2.

In an eleventh embodiment o, r, s, t, v, w, x and y are each 1.

The present invention also provides diagnostic agents comprising acompound described in any of the foregoing aspects and embodiments; andan imaging agent bound to the at least one chelator moiety. In someembodiments, the imaging agent is an echogenic substance, an opticalreporter, a boron neutron absorber, a paramagnetic metal ion, aferromagnetic metal, a gamma-emitting radioisotope, a positron-emittingradioisotope, or an x-ray absorber. In one set of embodiments, theimaging agent is a paramagnetic metal ion. In a particular embodiment,the paramagnetic metal ion is Gd(III). In another set of embodiments,the imaging agent is a gamma-emitting radioisotope or positron-emittingradioisotope selected from ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, and ¹⁵³Gd.

In one embodiment, the diagnostic agent has the structure,

In another embodiment, the diagnostic agent has the structure,

In another embodiment, the diagnostic agent has the structure,

In another embodiment, the diagnostic agent has the structure,

In another embodiment, the diagnostic agent has the structure,

In another embodiment, the diagnostic agent has the structure,

In a second aspect the present disclosure provides a diagnostic agentcomprising:

a. a compound of Formula (I-B)

or a pharmaceutically acceptable salt thereof; wherein

A is a D-amino acid residue or a peptide consisting of a D-amino acidresidue and a second D-amino acid;

D¹ and D² are independently selected from hydrogen and a chelatormoiety;

L¹ is a linker; or

L¹ and D², together with the nitrogen atom to which they are attached,form a five- to seven-membered ring; and

b. an imaging agent bound to the diagnostic agent.

In some embodiments, the imaging agent is bound to the diagnostic agentvia a chelator moiety.

In a first embodiment of the second aspect the imaging agent is anechogenic substance, an optical reporter, a boron neutron absorber, aparamagnetic metal ion, a ferromagnetic metal, a gamma-emittingradioisotope, a positron-emitting radioisotope, or an x-ray absorber. Ina second embodiment of the second aspect the imaging agent is aparamagnetic metal ion. In a third embodiment of the second aspect theparamagnetic metal ion is Gd(III).

In a fourth embodiment of the second aspect the imaging agent is agamma-emitting radioisotope or positron-emitting radioisotope selectedfrom ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, and ¹⁵³Gd.

In a third aspect the present disclosure provides a compound selectedfrom

or a pharmaceutically acceptable salt thereof.

In a fourth aspect the present disclosure provides a compound selectedfrom

In a fifth aspect the present disclosure provides a method of detecting,imaging, and/or monitoring elastin rich tissues in a patient comprisingthe steps of:

a. administering to the patient a diagnostic agent comprising:

-   -   1. a compound of Formula (I-B)

or a pharmaceutically acceptable salt thereof; wherein

A is a D-amino acid residue or a peptide consisting of a D-amino acidresidue and a second D-amino acid;

D¹ and D² are independently selected from hydrogen and a chelatormoiety;

L¹ is a linker; or

L¹ and D¹, together with the nitrogen atom to which they are attached,form a five- to seven-membered ring; and

-   -   2. an imaging agent; and

b. acquiring an image of a site of concentration of the compound in thepatient by a diagnostic imaging technique.

In a first embodiment of the fifth aspect the elastin rich tissues arethe arterial wall, uterus, lung, skin, and/or ligaments.

In a sixth aspect the present disclosure provides a method of detecting,imaging, and/or monitoring the presence of coronary plaque, carotidplaque, iliac/femoral plaque, aortic plaque, renal artery plaque, plaqueof any arterial vessel, aneurism, vasculitis, other diseases of thearterial wall, and/or damage or structural changes in ligaments, uterus,lungs or skin in a patient comprising the steps of:

a. administering to the patient a diagnostic agent comprising:

-   -   1. a compound of Formula (I-B)

or a pharmaceutically acceptable salt thereof; wherein

A is a D-amino acid residue or a peptide consisting of a D-amino acidresidue and a second D-amino acid;

D¹ and D² are independently selected from hydrogen and a chelatormoiety;

L¹ is a linker; or

L¹ and D², together with the nitrogen atom to which they are attached,form a five- to seven-membered ring; and

-   -   2. an imaging agent; and

b. acquiring an image of a site of concentration of the compound in thepatient by a diagnostic imaging technique.

The present invention also provides compounds of Formula (V),

or a pharmaceutically acceptable salt thereof; wherein

R^(p) is a hydrogen or an α-amino protecting group;

R² and R³ can be the same or different and are hydrogen, alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; and

R⁴ is hydrogen, alkyl, alkylaryl, or alkylarylalkyl,

wherein each R², R³, and R⁴ is unsubstituted or substituted with one ormore of the following: alkyl, alkenyl, cycloalkyl, alkylaryl,alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy, alkoxyalkyl,alkoxycarbonyl, heteroalkyl, heterocyclyl, or heterocyclylalkyl,—NR¹⁹R²⁰, —SH, —S(Pg), —OH, —PR¹⁹R²⁰, —P(O)R²¹R²², —CO₂H, ═O, halo,trifluoromethyl, cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO,—CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴,—NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H,—SO₂R²⁴, —SR²⁴, —S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴,NO₂, —C(═O)NHOR²⁴, —C(═O)NHNR²⁴R²⁴, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,or a chelator moiety;

R¹⁹ and R²⁰ are each independently selected from hydrogen, C₁₋₁₀alkylsubstituted with 0-3 R²³, aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkylsubstituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkyl substituted with 0-3R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³, and heterocyclylsubstituted with 0-3 R²³.

R²¹ and R²² are each independently selected from —OH, C₁₋₁₀alkylsubstituted with 0-3 R²³, aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkylsubstituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkyl substituted with 0-3R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³, and heterocyclylsubstituted with 0-3 R²³;

each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHNR²⁴R²⁴, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl;

each R²⁴ is independently selected from hydrogen, alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; and

Pg is a thiol protecting group.

In some embodiments, each R²⁴ is independently hydrogen, C₁₋₆alkyl,phenyl, benzyl, or C₁₋₆ alkoxy.

In some embodiments, R^(p) is hydrogen, Boc, or Fmoc; and R⁴ ishydrogen, alkyl, or alkylarylalkyl, substituted with an amino group. Forexample, R⁴ can be

In one set of embodiments, the compound has a structure as in Formula(VI),

or a pharmaceutically acceptable salt thereof; wherein R², R³, and R⁴are defined herein.

In another set of embodiments, the compound has a structure as inFormula (VII),

or a pharmaceutically acceptable salt thereof; wherein R² and R³ aredefined herein.

In another set of embodiments, the compound has a structure as inFormula (VIII)

or a pharmaceutically acceptable salt thereof; wherein R⁴ is definedherein.

In one embodiment, the compound has the structure,

In another embodiment, the compound has the structure,

In any of the forgoing aspects and embodiments, an alkyl group may beC₁₋₂₀ alkyl, C₁₋₁₀ alkyl, C₁₋₆ alkyl, or C₁₋₅ alkyl; a cycloalkyl groupmay be C₁₋₁₆ cycloalkyl, C₃₋₁₄ cycloalkyl, C₃₋₁₀cycloalkyl, orC₃₋₆cycloalkyl; an alkylaryl group may be C₁₋₁₀ alkyl-C₆₋₁₀aryl; analkenyl group may be C₂₋₄ alkenyl; an aryl group may be C₆₋₁₀ aryl; anarylalkyl group may be C₆₋₁₀aryl-C₁₋₁₀alkyl; an alkoxy group may be C₁₋₆alkoxy; an alkoxyalkyl group may be C₂₋₆alkoxyalkyl; a heterocyclylgroup may be a five-, six-, or seven-membered ring; and aheterocyclylalkyl group may be a heterocyclyl-C₁₋₁₀alkyl.

In any of the forgoing aspects and embodiments, the pharmaceuticallyacceptable salt may be any salt listed on pages 27-28 of the disclosure,or otherwise disclosed herein.

In any of the forgoing aspects and embodiments, the diagnostic agent maybe provided in the absence of a counterion (e.g., as a free base).

The present invention also provides methods for synthesizing any of theforegoing compounds according to the methods described herein. In someembodiments, the method may comprise reacting a compound with an imagingagent to form a diagnostic agent. In another embodiment, the method maycomprise reacting an intermediate molecule to produce a compound of theinvention. In some embodiments, the method may further compriseisolating and/or purifying the compound and/or diagnostic agent. Themethod may also comprise characterization of the compound and/ordiagnostic agent.

The present invention also provides methods of treating a patient. Themethod may comprise the steps of administering to the patient adiagnostic agent as in any foregoing diagnostic agent embodiments; andacquiring an image of a site of concentration of the diagnostic agent inthe patient by a diagnostic imaging technique. In some embodiments, thetreating may comprise detecting, imaging, and/or monitoring elastin-richtissues in a patient. The elastin-rich tissues may be located within thearterial wall, uterus, lung, skin, and/or ligaments. In someembodiments, the treating may comprise detecting, imaging, and/ormonitoring the presence and/or amount of coronary plaque, carotidplaque, iliac/femoral plaque, aortic plaque, renal artery plaque, plaqueof any arterial vessel, aneurism, vasculitis, other diseases of thearterial wall, and/or damage or structural changes in ligaments, uterus,lungs or skin in a patient.

Other aspects of the invention may include suitable combinations ofembodiments and aspects disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows transaxial MR images of rabbit abdominal aorta using animaging agent described herein.

Other aspects, embodiments and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying FIGURES areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every FIGURE, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION

The present disclosure is directed to compounds, diagnostic agents, andrelated methods. In some embodiments, methods for synthesizing compoundsand/or diagnostic agents are provided. In some embodiments, methods fortreating a patient are provided. For example, compounds, diagnosticagents, compositions, and kits for detecting and/or imaging and/ormonitoring a pathological disorder associated with coronary plaque,carotid plaque, iliac/femoral plaque, aortic plaque, renal arteryplaque, plaque of the arterial vessel, aneurism, vasculitis, otherdiseases of the arterial wall, and/or damage or structural changes inligaments, uterus, lungs or skin, are provided. In addition, thedisclosure provides methods of detecting and/or imaging and/ormonitoring changes in the arterial wall, including expansive andconstrictive remodeling, total vessel wall area, internal lumen size,and exterior arterial perimeter. Other aspects and embodiments may befound in the description provided herein.

Unless otherwise specifically noted herein, the terms set forth belowwill have the following definitions.

In some instances, the number of carbon atoms in any particular group isdenoted before the recitation of the group. For example, the term “C₆₋₁₀aryl” denotes an aryl group containing from six to ten carbon atoms, andthe term “C₆₋₁₀ aryl-C₁₋₁₀ alkyl,” refers to an aryl group of six to tencarbon atoms attached to the parent molecular moiety through an alkylgroup of one to ten carbon atoms. Where these designations exist theysupersede all other definitions contained herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise.

The term “alkenyl,” as used herein, refers to a straight or branchedchain hydrocarbon of two to fourteen carbon atoms containing at leastone carbon-carbon double bond.

The term “alkenylene,” as used herein, refers to a divalent groupderived from a straight or branched chain hydrocarbon of two to fourteencarbon atoms containing at least one carbon-carbon double bond.

The term “alkoxy,” as used herein refers to an alkyl group attached tothe parent molecular moiety through an oxygen atom.

The term “alkoxyalkyl,” as used herein, refers to an alkoxy groupattached to the parent molecular moiety through an alkyl group.

The term “alkoxycarbonyl,” as used herein, refers to an alkoxy groupattached to the parent molecular moiety through a carbonyl group.

The term “alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon.

The term “alkylaryl,” as used herein, refers to an alkyl group attachedto the parent molecular moiety through an aryl group.

The term “alkylcarbonyl,” as used herein, refers to an alkyl groupattached to the parent molecular moiety through a carbonyl group.

The term “alkylene,” as used herein, refers to a divalent group derivedfrom a straight or branched chain saturated hydrocarbon of one tofourteen carbon atoms.

As used herein, the phrase “amino acid residue” means a moiety derivedfrom a naturally-occurring or synthetic organic compound containing anamino group (—NH₂), a carboxylic acid group (—COOH), and any of variousside groups, especially any of the 20 compounds that have the basicformula NH₂CHRCOOH, and that link together by peptide bonds to formproteins or that function as chemical messengers and as intermediates inmetabolism. For example, in compound X

the portion of the molecule denoted as “A” is a residue of the aminoacid D-leucine.

The term “aryl,” as used herein, refers to a phenyl group, or a bicyclicfused ring system wherein one or more of the rings is a phenyl group.Bicyclic fused ring systems consist of a phenyl group fused to amonocyclic cycloalkenyl group, a monocyclic cycloalkyl group, or anotherphenyl group. The aryl groups of the present invention can be attachedto the parent molecular moiety through any substitutable carbon atom inthe group. Representative examples of aryl groups include, but are notlimited to, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl,naphthyl, phenyl, and tetrahydronaphthyl.

The term “arylalkyl,” as used herein, refers to an aryl group attachedto the parent molecular moiety through an alkyl group.

The term “arylalkylene,” as used herein, refers to a divalent arylalkylgroup, where one point of attachment to the parent molecular moiety ison the aryl portion and the other is on the alkyl portion.

The term “alkylarylalkyl,” as used herein, refers to an alkylaryl groupattached to the parent molecular moiety through an alkyl group.

The term “arylene,” as used herein, refers to a divalent aryl group.

The term “cycloalkyl,” as used herein, refers to a saturated monocyclic,bicyclic, or tricyclic hydrocarbon ring system having three to fourteencarbon atoms and zero heteroatoms. Representative examples of cycloalkylgroups include, but are not limited to, cyclopropyl, cyclopentyl,bicyclo[3.1.1]heptyl, and adamantyl.

The term “cycloalkylene,” as used herein, refers to a divalentcycloalkyl group.

The term “cycloalkylmethyl,” as used herein, refers to a cycloalkylgroup attached to the parent molecular moiety through a —CH₂— group.

The term “heteroalkyl,” as used herein, refers to an alkyl group whereinone to seven of the carbon atoms are replaced by a heteroatom selectedfrom O, NH, and S.

The term “heteroalkylene,” as used herein, refers to an alkylene groupwherein one to seven of the carbon atoms are replaced by a heteroatomselected from O, NH, and S.

The term “heterocyclyl,” as used herein, refers to a five-, six-, orseven-membered ring containing one, two, or three heteroatomsindependently selected from the group consisting of nitrogen, oxygen,and sulfur. The five-membered ring has zero to two double bonds and thesix- and seven-membered rings have zero to three double bonds. The term“heterocyclyl” also includes bicyclic groups in which the heterocyclylring is fused to a phenyl group, a monocyclic cycloalkenyl group, amonocyclic cycloalkyl group, or another monocyclic heterocyclyl group.The heterocyclyl groups of the present invention can be attached to theparent molecular moiety through a carbon atom or a nitrogen atom in thegroup. Examples of heterocyclyl groups include, but are not limited to,benzothienyl, furyl, imidazolyl, indolinyl, indolyl, isothiazolyl,isoxazolyl, morpholinyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl,pyridinyl, pyrrolidinyl, pyrrolopyridinyl, pyrrolyl, thiazolyl, thienyl,and thiomorpholinyl.

The term “heterocyclylalkyl,” as used herein, refers to a heterocyclylgroup attached to the parent molecular moiety through an alkyl group.

The term “heterocyclylalkylene,” as used herein, refers to a divalentheterocyclylalkyl group, where one point of attachment to the parentmolecular moiety is on the heterocyclyl portion and the other is on thealkyl portion.

The term “heterocyclylene,” as used herein, refers to a divalentheterocyclyl group.

The term “halo,” as used herein, refers to Br, Cl, F, or I.

The term “carbonyl,” as used herein, refers to —C(O)—.

The term “cyano,” as used herein, refers to —CN.

The term “amino,” as used herein, refers to —NR¹⁹R²⁰, wherein R¹⁹ andR²⁰ are defined herein.

As used herein, the phrase “donor atom” refers to the atom directlyattached to a metal by a chemical bond.

The term “linker,” as used herein, refers to a portion of a moleculethat serves as a spacer between two other portions of the molecule.Linkers may also serve other functions as described herein.

The terms “chelator” and “chelator moiety,” as used herein, refer to themoiety or group on a molecule that binds to a metal ion through one ormore donor atoms. The chelator is optionally attached to the parentmolecular moiety through a linker, L². Examples of suitable L² groupsinclude, but are not limited to, —C(O)CH₂—Ar—CH₂NHC(O)—, where Ar is anarylene group; —C(O)—; —C(O)—Het-NHNHC(O)—, where Het is heteroarylene;—CH₂—Ar—CH₂—, where Ar is an arylene group; —C(O)—Het-; as well as othergroups disclosed herein. In certain embodiments of the compounds and/ordiagnostic agents of the disclosure, the chelator is a surfactantcapable of forming an echogenic substance-filled lipid sphere ormicrobubble.

In certain other embodiments, the chelator moiety has a formula selectedfrom

wherein

each A¹ is independently selected from —NR¹⁹R²⁰, —N(R²⁶)₂, —SH, —S(Pg),—OH, —PR¹⁹R²⁰, —P(O)R²¹R²², —CO₂H, a bond to the parent molecularmoiety, and a bond to L²;

each A² is independently selected from N(R²⁶), N(R¹⁹), S, O, P(R¹⁹), and—OP(O)(R²¹)O—;

A³ is N;

A⁴ is selected from OH and OC(═O)C₁₋₂₀ alkyl;

A⁵ is OC(═O)C₁₋₂₀ alkyl;

each E is independently selected from C₁₋₁₆alkylene substituted with 0-3R²³, C₆₋₁₀arylene substituted with 0-3 R²³, C₃₋₁₀cycloalkylenesubstituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkylene substituted with0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylene substituted with 0-3 R²³,C₁₋₁₀alkyl-C₆₋₁₀arylene substituted with 0-3 R²³, and heterocyclylenesubstituted with 0-3 R²³;

E¹ is selected from a bond and E;

each E² is independently selected from C₁₋₁₆alkyl substituted with 0-3R²³, C₆₋₁₀aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substitutedwith 0-3 R²³, heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³,C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₁₋₁₀alkyl-C₆₋₁₀arylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;

E³ is C₁₋₁₀alkylene substituted with 1-3 R³²;

Pg is a thiol protecting group;

R¹⁹ and R²⁰ are each independently selected from a bond to L², a bond tothe parent molecular moiety, hydrogen, C₁₋₁₀alkyl substituted with 0-3R²³, aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substituted with0-3, heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³,C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³, and heterocyclylsubstituted with 0-3 R²³.

R²¹ and R²² are each independently selected from a bond L², a bond tothe parent molecular moiety, —OH, C₁₋₁₀alkyl substituted with 0-3 R²³,aryl substituted with 0-3 R²³. C₃₋₁₀cycloalkyl substituted with 0-3 R²³,heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;

each R²³ is independently selected from a bond to L², a bond to theparent molecular moiety, ═O, halo, trifluoromethyl, cyano, —CO₂R²⁴,—C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴,—OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂,—NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴, —S(═O)R²⁴,—SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂, —C(═O)NHOR²⁴,—C(═O)NHNR²⁴R²⁴, —OCH₂CO₂H, 2-(1-morpholino)ethoxy, C₁₋₅alkyl,C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl, C₂₋₆alkoxyalkyl, arylsubstituted with 0-2 R²⁴, and heterocyclyl;

each R²⁴ is independently selected from a bond to L², a bond to theparent molecular moiety, hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl;

each R²⁶ is independently a coordinate bond to a metal or a hydrazineprotecting group;

each R³² selected from R³⁴, ═O, —CO₂R³³, —C(═O)R³³, —C(═O)N(R³³)₂,—CH₂OR³³, —OR³³, —N(R³³)₂, and C₂-C₄ alkenyl;

each R³³ is independently selected from R³⁴, hydrogen, C₁-C₆ alkyl,phenyl, benzyl, and trifluoromethyl; and

R³⁴ is a bond to L²;

wherein at least one of A¹, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, and R³⁴ is abond to L² or the parent molecular moiety.

In some embodiments, each R²⁴ is independently hydrogen, C₁₋₆alkyl,phenyl, benzyl, or C₁₋₆ alkoxy.

In an embodiment of the present disclosure, the chelant is of theformula:

wherein

A^(1c) is a bond to L²;

A^(1a), A^(1b), A^(1d) and A^(1e) are each —CO₂H;

A^(3a), A^(3b), and A^(3c) are each N;

E^(b), and E^(c) are C₂alkylene; and

E^(a), E^(d), E^(e), E^(f), and E^(g) are CH₂.

In another embodiment of the present disclosure the chelant is of theformula:

wherein:

A^(3a), A^(3b), A^(3c) and A^(3d) are each N;

A^(1a) is a bond to L²;

A^(1b), A^(1c) and A^(1d) are each —CO₂H;

E^(a), E^(c), E^(g) and E^(e) are each CH₂; and

E^(b), E^(d), E^(f) and E^(h) are each C₂alkylene.

In another embodiment of the present disclosure, the chelant is of theformula:

wherein

A^(1a) is —N(R²⁶)₂;

A^(1b) is NHR¹⁹;

E is a bond;

R¹⁹ is a bond to L²; and

each R²⁶ is a co-ordinate bond to a metal.

In some embodiments, the chelator moiety comprises one of the followingstructures,

As used herein, the terms “ancillary” and “co-ligands” refers to ligandsthat serve to complete the coordination sphere of the radionuclidetogether with the chelator of the reagent. For radiopharmaceuticalscomprising a binary ligand system, the radionuclide coordination spherecomprises one or more chelators from one or more reagents and one ormore ancillary or co-ligands, provided that there are a total of twotypes of ligands or chelators. For example, a radiopharmaceuticalcomprised of one chelator from one reagent and two of the same ancillaryor co-ligands and a radiopharmaceutical comprising two chelators fromone or two reagents and one ancillary or co-ligand are both consideredto comprise binary ligand systems. For radiopharmaceuticals comprising aternary ligand system, the radionuclide coordination sphere comprisesone or more chelators from one or more reagents and one or more of twodifferent types of ancillary or co-ligands, provided that there are atotal of three types of ligands or chelators. For example, aradiopharmaceutical comprised of one chelator from one reagent and twodifferent ancillary or co-ligands is considered to comprise a ternaryligand system.

Ancillary or co-ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals comprise one or more oxygen, nitrogen,carbon, sulfur, phosphorus, arsenic, selenium, and tellurium donoratoms. A ligand can be a transfer ligand in the synthesis of aradiopharmaceutical and also serve as an ancillary or co-ligand inanother radiopharmaceutical. Whether a ligand is termed a transfer orancillary or co-ligand depends on whether the ligand remains in theradionuclide coordination sphere in the radiopharmaceutical, which isdetermined by the coordination chemistry of the radionuclide and thechelator of the reagent or reagents.

As used herein, the term “diagnostic agent” refers to a compound thatmay be used to detect, image and/or monitor the presence and/orprogression of a condition(s), pathological disorder(s) and/ordisease(s). It should be understood that all compounds of the presentinvention that contain an imaging agent are diagnostic agents. Forexample, a compound of Formula (I-A) wherein one of D¹ and D² is animaging agent is a diagnostic agent.

The term “diagnostic imaging technique,” as used herein, refers to aprocedure used to detect a diagnostic agent.

The terms “diagnostic kit” and “kit”, as used herein, refer to acollection of components in one or more vials that are used by thepracticing end user in a clinical or pharmacy setting to synthesizediagnostic agents. The kit provides all the requisite components tosynthesize and use the diagnostic agents (except those that are commonlyavailable to the practicing end user such as water or saline forinjection), such as a solution of the imaging agent or a precursorthereof, equipment for heating during the synthesis of the diagnosticagent, equipment necessary for administering the diagnostic agent to thepatient such as syringes and shielding (if required), and imagingequipment.

The term “imaging moiety,” as used herein, refers to a portion orportions of a molecule that contain an imaging agent. The term “imagingagent,” as used herein, refers to an element or functional group in adiagnostic agent that allows for the detection, imaging, and/ormonitoring of the presence and/or progression of a condition(s),pathological disorder(s), and/or disease(s). The imaging agent may bebound to the diagnostic agent via a bond, such as a covalent bond, anionic bond, a hydrogen bond, a dative bond (e.g. complexation orchelation between metal ions and monodentate or multidentate ligands),or the like. For example, the imaging agent may be a paramagnetic metalion bound to the diagnostic agent by chelation of the metal ion to amonodentate or multidentate ligand (e.g., chelating moiety) of thediagnostic agent. The imaging moiety may contain a linker, L³, whichconnects the imaging agent to the parent molecular moiety. Examples ofsuitable L³ groups include straight or branched chain alkylene groups,—C(O)—, and the like.

The imaging agent may be an echogenic substance (either liquid or gas),non-metallic isotope, an optical reporter, a boron neutron absorber, aparamagnetic metal ion, a ferromagnetic metal, a gamma-emittingradioisotope, a positron-emitting radioisotope, or an x-ray absorber.

Suitable echogenic gases include a sulfur hexafluoride orperfluorocarbon gas, such as perfluoromethane, perfluoroethane,perfluoropropane, perfluorobutane, perfluorocyclobutane,perfluoropentane, or perfluorohexane.

Suitable non-metallic isotopes include ¹¹C, ¹⁴C, ¹³N, ¹⁸F, ¹²³I, ¹²⁴I,and ¹²⁵I.

Suitable optical reporters include a fluorescent reporter andchemiluminescent groups.

Suitable radioisotopes include ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga,⁶⁸Ga, and ¹⁵³Gd. In a specific embodiment of the present disclosuresuitable radioisotopes include ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, and ¹⁵³Gd.

Suitable paramagnetic metal ions include: Gd(III), Dy(III), Fe(III), andMn(II).

Suitable X-ray absorbers include: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La,Au, Yb, Dy, Cu, Rh, Ag, Ir and I.

As used herein, the term “metallopharmaceutical” means a pharmaceuticalcomprising a metal. The metal is the origin of the imagable signal indiagnostic applications and the source of the cytotoxic radiation inradiotherapeutic applications.

The term “radiopharmaceutical,” as used herein, refers to ametallopharmaceutical in which the metal is a radioisotope.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, diagnostic agents, materials, compositions, and/or dosageforms that are, within the scope of sound medical judgment, suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The compounds and/or diagnostic agents of the present disclosure canexist as pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salt,” as used herein, represents salts or zwitterionic formsof the compounds and/or diagnostic agents of the present disclosurewhich are water or oil-soluble or dispersible, which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of patients without excessive toxicity, irritation, allergicresponse, or other problem or complication commensurate with areasonable benefit/risk ratio, and are effective for their intended useThe salts can be prepared during the final isolation and purification ofthe compounds and/or diagnostic agents or separately by reacting asuitable nitrogen atom with a suitable acid. Representative acidaddition salts include acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate; digluconate, glycerophosphate, hemisulfate,heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate,3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate, and undecanoate. Examples of acids which can beemployed to form pharmaceutically acceptable addition salts includeinorganic acids such as hydrochloric, hydrobromic, sulfuric, andphosphoric, and organic acids such as oxalic, maleic, succinic, andcitric.

Basic addition salts can be prepared during the final isolation andpurification of the compounds and/or diagnostic agents by reacting acarboxy group with a suitable base such as the hydroxide, carbonate, orbicarbonate of a metal cation or with ammonia or an organic primary,secondary, or tertiary amine. The cations of pharmaceutically acceptablesalts include lithium, sodium, potassium, calcium, magnesium, andaluminum, as well as nontoxic quaternary amine cations such as ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine,pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,and N,N′-dibenzylethylenediamine. Other representative organic aminesuseful for the formation of base addition salts include ethylenediamine,ethanolamine, diethanolamine, meglumine, piperidine, and piperazine.

In some embodiments, the compounds and/or diagnostic agents describedherein may be provided in the absence of a counterion (e.g., as a freebase).

As used herein, the term “reagent” means a compound of this disclosurecapable of direct transformation into a diagnostic agent of thisdisclosure. Reagents may be utilized directly for the preparation of thediagnostic agents of this disclosure or may be a component in a kit ofthis disclosure.

As used herein, the term “lyophilization aid” means a component that hasfavorable physical properties for lyophilization, such as the glasstransition temperature, and is added to the formulation to improve thephysical properties of the combination of all the components of theformulation for lyophilization.

As used herein, the phrase “solubilization aid” is a component thatimproves the solubility of one or more other components in the mediumrequired for the formulation.

As used herein, the phrase “stabilization aid” means a component that isadded to the metallopharmaceutical or to the diagnostic kit either tostabilize the metallopharmaceutical or to prolong the shelf-life of thekit before it must be used. Stabilization aids can be antioxidants,reducing agents or radical scavengers and can provide improved stabilityby reacting with species that degrade other components or themetallopharmaceutical.

The term “stable”, as used herein, refers to compounds and/or diagnosticagents which possess the ability to allow manufacture and which maintaintheir integrity for a sufficient period of time to be useful for thepurposes detailed herein. Typically, the compounds and/or diagnosticagents of the present disclosure are stable at a temperature of 40° C.or less in the absence of moisture or other chemically reactiveconditions for at least a week.

The term “buffer,” as used herein, refers to a substance used tomaintain the pH of the reaction mixture from about 3 to about 10.

The term “sterile,” as used herein, means free of or using methods tokeep free of pathological microorganisms.

As used herein, the term “bacteriostat” means a component that inhibitsthe growth of bacteria in a formulation either during its storage beforeuse of after a diagnostic kit is used to synthesize a diagnostic agent.

The term “carrier”, as used herein, refers to an adjuvant or vehiclethat may be administered to a patient, together with the compoundsand/or diagnostic agents of this disclosure which does not destroy theactivity thereof and is non-toxic when administered in doses sufficientto deliver an effective amount of the diagnostic agent and/or compound.

Asymmetric centers exist in the compounds and/or diagnostic agents ofthe present invention. These centers are designated by the symbols “R”or “S”, depending on the configuration of substituents around the chiralcarbon atom. It should be understood that the invention encompasses allstereochemical isomeric forms of the present compounds and/or diagnosticagents, or mixtures thereof, unless otherwise specifically stated.Individual stereoisomers of compounds and/or diagnostic agents can beprepared synthetically from commercially available starting materialswhich contain chiral centers or by preparation of mixtures ofenantiomeric products followed by separation such as conversion to amixture of diastereomers followed by separation or recrystallization,chromatographic techniques, or direct separation of enantiomers onchiral chromatographic columns. Starting compounds of particularstereochemistry are either commercially available or can be made andresolved by techniques known in the art.

Certain compounds and/or diagnostic agents of the present disclosure mayalso exist in different stable conformational forms which may beseparable. Torsional asymmetry due to restricted rotation about anasymmetric single bond, for example because of steric hindrance or ringstrain, may permit separation of different conformers. The presentdisclosure includes each conformational isomer of these compounds and/ordiagnostic agents and mixtures thereof.

When any variable occurs more than one time in any substituent or in anyformula, its definition on each occurrence is independent of itsdefinition at every other occurrence. Thus, for example, if a group isshown to be substituted with 0-2 R²³, then said group may optionally besubstituted with up to two R²³, and R²³ at each occurrence is selectedindependently from the defined list of possible R²³. Also, by way ofexample, for the group —N(R²⁴)₂, each of the two R²⁴ substituents on thenitrogen is independently selected from the defined list of possibleR²⁴. Combinations of substituents and/or variables are permissible onlyif such combinations result in stable compounds and/or diagnosticagents. When a bond to a substituent is shown to cross the bondconnecting two atoms in a ring, then such substituent may be bonded toany atom on the ring.

When the imaging agent is a radioisotope, the compound may furthercomprise a first ancillary ligand and a second ancillary ligand capableof stabilizing the radioisotope. A large number of ligands can serve asancillary or co-ligands, the choice of which is determined by a varietyof considerations such as the ease of synthesis of theradiopharmaceutical, the chemical and physical properties of theancillary ligand, the rate of formation, the yield, and the number ofisomeric forms of the resulting radiopharmaceuticals, the ability toadminister said ancillary or co-ligand to a patient without adversephysiological consequences to said patient, and the compatibility of theligand in a lyophilized kit formulation. The charge and lipophilicity ofthe ancillary ligand will affect the charge and lipophilicity of theradiopharmaceutical. For example, the use of4,5-dihydroxy-1,3-benzenedisulfonate results in radiopharmaceuticalswith an additional two anionic groups because the sulfonate groups willbe anionic under physiological conditions. The use of N-alkylsubstituted 3,4-hydroxypyridinones results in radiopharmaceuticals withvarying degrees of lipophilicity depending on the size of the alkylsubstituents.

It should also be understood that the compounds and/or diagnostic agentsof this disclosure may adopt a variety of conformational and ionic formsin solution, in pharmaceutical compositions and in vivo. Although thedepictions herein of specific compounds and/or diagnostic agents of thisdisclosure are of particular conformations and ionic forms, otherconformations and ionic forms of those compounds and/or diagnosticagents are envisioned and embraced by those depictions.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this disclosure include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, TRIS(tris(hydroxymethyl)amino-methane), partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes, such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances,polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycoland wool fat.

According to this disclosure, the pharmaceutical compositions may be inthe form of a sterile injectable preparation, for example a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant.

In some cases, depending on the dose and rate of injection, the bindingsites on plasma proteins may become saturated with prodrug and activatedagent. This leads to a decreased fraction of protein-bound agent andcould compromise its half-life or tolerability as well as theeffectiveness of the agent. In these circumstances, it is desirable toinject the prodrug agent in conjunction with a sterile albumin or plasmareplacement solution. Alternatively, an apparatus/syringe can be usedthat contains the contrast agent and mixes it with blood drawn up intothe syringe; this is then re-injected into the patient.

The compounds, diagnostic agents and pharmaceutical compositions of thepresent disclosure may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsand vehicles. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

When administered orally, the pharmaceutical compositions of thisdisclosure may be administered in any orally acceptable dosage formincluding, but not limited to, capsules, tablets, aqueous suspensions orsolutions. In the case of tablets for oral use, carriers that arecommonly used include lactose and corn starch. Lubricating agents, suchas magnesium stearate, are also typically added. For oral administrationin a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

Alternatively, when administered in the form of suppositories for rectaladministration, the pharmaceutical compositions of this disclosure maybe prepared by mixing the agent with a suitable non-irritating excipientthat is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

As noted before, the pharmaceutical compositions of this disclosure mayalso be administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds and/or diagnostic agents of thisdisclosure include, but are not limited to, mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active components suspended or dissolved in one ormore pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,typically, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

For administration by nasal aerosol or inhalation, the pharmaceuticalcompositions of this disclosure are prepared according to techniqueswell-known in the art of pharmaceutical formulation and may be preparedas solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 5% to about 95% active compound(w/w). Typically, such preparations contain from about 20% to about 80%active compound.

For intravenous and other types of administration, acceptable doseranges range from about 0.001 to about 1.0 mmol/kg of body weight, withthe typical dose of the active ingredient compound ranging from about0.001 to about 0.5 mmol/kg of body weight. Even more typical is fromabout 0.01 to about 0.1 mmol/kg, and the most typical dose of the activeingredient compound is from about 0.0001 and to about 0.05 mmol/kg.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage regimens for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration, rate ofexcretion, drug combination and the judgment of the treating physician.

Buffers useful in the preparation of diagnostic agents and kits thereofinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids useful in the preparation of diagnostic agents andkits thereof include but are not limited to mannitol, lactose, sorbitol,dextran, Ficoll, and polyvinylpyrrolidine (PVP).

Stabilization aids useful in the preparation of diagnostic agents andkits thereof include but are not limited to ascorbic acid, cysteine,monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid,and inositol.

Solubilization aids useful in the preparation of diagnostic agents andkits thereof include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polyoxyethylene sorbitanmonooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics) and lecithin. Typical solubilizing aids are polyethyleneglycol, and Pluronics copolymers.

Bacteriostats useful in the preparation of diagnostic agents and kitsthereof include but are not limited to benzyl alcohol, benzalkoniumchloride, chlorbutanol, and methyl, propyl or butyl paraben.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer can alsoserve as a transfer ligand, a lyophilization aid can also serve as atransfer, ancillary or coligand and so forth.

The predetermined amounts of each component in the formulation aredetermined by a variety of considerations that are in some casesspecific for that component and in other cases dependent on the amountof another component or the presence and amount of an optionalcomponent. In general, the minimal amount of each component is used thatwill give the desired effect of the formulation. The desired effect ofthe formulation is that the practicing end user can synthesize thediagnostic agent and have a high degree of certainty that the diagnosticagent can be injected safely into a patient and will provide diagnosticinformation about the disease state of that patient.

The diagnostic kits of the present disclosure can also contain writteninstructions for the practicing end user to follow to synthesize thediagnostic agents. These instructions may be affixed to one or more ofthe vials or to the container in which the vial or vials are packagedfor shipping or may be a separate insert, termed the package insert.

X-ray contrast agents, ultrasound contrast agents andmetallopharmaceuticals for use as magnetic resonance imaging contrastagents are provided to the end user in their final form in a formulationcontained typically in one vial, as either a lyophilized solid or anaqueous solution. The end user reconstitutes the lyophilized solid withwater or saline and withdraws the patient dose or simply withdraws thedose from the aqueous solution formulation as provided.

These diagnostic agents, whether for gamma scintigraphy, positronemission tomography, MRI, ultrasound or x-ray image enhancement, areuseful, inter alia, to detect and monitor changes in cardiovasculardiseases over time.

Methods for synthesizing the compounds and diagnostic agents describedherein are also provided. In some cases, the method may comprisereacting a compound and/or intermediate described herein, to produce acompound and/or diagnostic agent of the invention. For example, themethod may comprise reacting a compound with an imaging agent to form adiagnostic agent, as described herein. In another example, the methodmay comprise reacting an intermediate molecule to produce a compound ofthe invention. In some cases, the intermediate molecule may be acompound comprising a hydroxylamine derivative, a hydroxamic acid, ahydroxamate ester, and amine, or the like. Other intermediate moleculesare described herein, including the Examples. The method may furthercomprise isolating and/or purifying the compound and/or diagnosticagent, for example, by chromatography (e.g., column chromatography,HPLC), crystallization, filtration, solvent extraction, and the like.The method may also comprise characterization of the compound and/ordiagnostic agent by mass spectrometry, NMR, and the like.

The compounds and/or diagnostic agents of the present disclosure can beprepared following the procedures described herein. In some cases, thecompound and/or diagnostic agent may be synthesized by coupling ahydroxylamine derivative with a carbonyl group such as a carboxylicacid, acyl halide, ester, or the like, to form a hydroxamate ester. Forexample, Scheme 1 shows the condensation of a carboxylic acid moietywith a hydroxylamine derivative (e.g., H₂NOR⁴) to form the hydroxamateester. In some cases, the hydroxylamine derivative may be substitutedwith a chelator moiety.

In some embodiments, the compound and/or diagnostic agent may besynthesized by coupling hydroxylamine with a carbonyl group to form ahydroxamic acid, which may be further substituted with, for example, achelator moiety. As shown in Scheme 2, reaction of a carboxylic estermoiety with hydroxylamine forms a hydroxyamic acid, which is thensubstituted at the oxygen with a species comprising a leaving group,i.e., Y—R⁴, wherein Y is a leaving group and R⁴ comprises a chelatormoiety.

Compounds and diagnostic agents described herein may also be synthesizedusing various methods known in the art to form carbon-carbon bonds,carbon-heteroatom bonds, and the like. For example, portions of thecompounds and diagnostic agents may be bonded to one another via amino,ether, thioether, ester, thioester, amide, thiourea, or other linkages.In some cases, the chelator moiety may be bonded to the compound ordiagnostic agent via an amide linkage.

Those of ordinary skill in the art would be able to select suitablemethods for synthesizing a compound or diagnostic agent having aparticular linkage. For example, methods for coupling amino acids orpeptides, as described more fully below, may be used in the context ofthe invention to form an amide linkage between portions of the compoundor diagnostic agent. In some cases, alkylation of an alcohol or a thiolmay be used to form an ether or a thioether, respectively. For example,reaction of a thiol with an alkyl species comprising a leaving group(e.g., halo, tosyl, mesyl, or the like) may result in formation of abond between the thioether and the alkyl group, i.e., a thioether. Insome embodiments, the compounds or diagnostic agent may include athiourea linkage, which can be formed using various methods known in theart, including an acylation reaction between an amine moiety and aisothiocyanate moiety.

In some cases, the Mitsunobu reaction may be utilized to form a wideranges of linkages, including esters, phenyl ethers, thioethers, andothers, by reaction of a nucleophile (e.g., an acidic nucleophile) witha primary or secondary alcohol in the presence ofdiethylazodicarboxylate (DEAD). Those of ordinary skill in the art wouldbe able to select the appropriate nucleophile suitable for use in aparticular application. For example, reaction between an alcohol and aphenol under Mitsunobu conditions may produce an aryl ether, whilereaction between an alcohol an a carboxylic acid or thiol underMitsunobu conditions may produce an ester or thioester, respectively.

Compounds and diagnostic agents described herein may also comprise aphosphonate ester linkage. In some embodiments, a phosphonate ester maybe synthesized by coupling of a phosphonic acid and an alcohol, forexample, in the presence of DEAD or dicyclocarbodiimide (DCC).Additional methods for synthesizing phosphonate esters are described in,for example, Savignac, P. et al., Modern Phosphonate Chemistry, CRCPress: New York, 2003, the contents of which are incorporated herein byreference.

Other methods for forming carbon-carbon bonds may be used to synthesizecompounds or diagnostic agents described herein, such as olefinmetathesis. As used herein, “metathesis” or “olefin metathesis” is givenits ordinary meaning in the art and refers to a chemical reaction inwhich two reacting species exchange partners in the presence of atransition-metal catalyst, according to the formula shown in Scheme 3,forming a carbon-carbon double bond between the two reacting species andethylene as a byproduct. Examples of different kinds of metathesisreactions including cross metathesis, ring-closing metathesis,ring-opening metathesis, acyclic diene metathesis, alkyne metathesis,enyne metathesis, and the like. Typically, metathesis reactions areperformed in the presence of a metathesis catalyst, which may compriseruthenium, molybdenum, or tungsten (e.g., Grubbs' 1^(st) generationcatalyst, Grubbs' 2nd generation catalyst, Schrock's catalyst).

Metal-catalyzed cross-coupling reactions may also be used in thesynthesis of compounds and diagnostic agents. For example, aryl halidesmay be reacted with various species in the presence of a metal catalystto form linkages including biaryl ethers, acetylenes, alkenylaryls(e.g., styrene and styrene derivatives), arenes, and the like. Examplesof cross-coupling reactions suitable for use in the context of theinvention include the Ullmann, Sonogashira/Castro-Stevens, Heck, Stille,Suzuki, and other related reactions. Those of ordinary still in the artwould be able to select the appropriate reactants, catalysts, andreaction conditions for synthesizing a particular desired compound ordiagnostic agent.

Cycloaddition chemistry may also be used to synthesize compounds anddiagnostic agents described herein. For example, “click” chemistry maybe utilized, wherein a [3+2] cycloaddition between an azide-containingspecies and an alkyne-containing species may form a triazole linkagebetween the two species. Such reactions may be performed under mildconditions and with high tolerance for a wide range of functionalgroups.

In some cases, the compound or diagnostic agent may include a peptide,polypeptide, and/or peptidomimetic, which may be synthesized usingvarious known methods. Generally, peptides, polypeptides andpeptidomimetics are elongated by deprotecting the alpha-amine of theC-terminal residue and coupling the next suitably protected amino acidthrough a peptide linkage using the methods described. This deprotectionand coupling procedure is repeated until the desired sequence isobtained. This coupling can be performed with the constituent aminoacids in a stepwise fashion, or condensation of fragments (two toseveral amino acids), or combination of both processes, or by solidphase peptide synthesis according to the method originally described inJ. Am. Chem. Soc., 1963, 85, 2149-2154.

The peptides, polypeptides and peptidomimetics may also be synthesizedusing automated synthesizing equipment. In addition to the foregoing,procedures for peptide, polypeptide and peptidomimetic synthesis aredescribed in Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed.,Pierce Chemical Co., Rockford, Ill. (1984); Gross, Meienhofer,Udenfriend, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1, 2,3, 5, and 9, Academic Press, New York, (1980-1987); Bodanszky, PeptideChemistry: A Practical Textbook, Springer-Verlag, New York (1988); andBodanszky et al., The Practice of Peptide Synthesis, Springer-Verlag,New York (1984).

The coupling between two amino acid derivatives, an amino acid and apeptide, polypeptide or peptidomimetic, two peptide, polypeptide orpeptidomimetic fragments, or the cyclization of a peptide, polypeptideor peptidomimetic can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic acid anhydride (isobutylchloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimides) method, activeester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method,Woodward reagent K method, carbonyldiimidazole method, phosphorusreagents such as BOP-Cl, or oxidation-reduction method. Some of thesemethods (especially the carbodiimide) can be enhanced by the addition of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole. These couplingreactions may be performed either in solution (liquid phase) or on asolid phase, such as polystyrene or a suitable resin (vide infra).

The functional groups of the constituent amino acids or amino acidmimetics are typically protected during the coupling reactions to avoidundesired bonds being formed. The protecting groups that can be used arelisted in Greene, Protective Groups in Organic Synthesis, John Wiley &Sons, New Jersey (2007) and The Peptides: Analysis, Synthesis, Biology,Vol. 3, Academic Press, New York (1981).

The α-carboxyl group of the C-terminal residue may be protected by anester that can be cleaved to give the carboxylic acid. These protectinggroups include:

(1) alkyl esters such as methyl and t-butyl;

(2) aryl esters such as benzyl and substituted benzyl, or

(3) esters that can be cleaved by mild base treatment or mild reductivemeans such as trichloroethyl and phenacyl esters.

In the solid phase case, the C-terminal amino acid is attached to aninsoluble carrier (usually polystyrene). These insoluble carrierscontain a group that will react with the carboxyl group to form a bondwhich is stable to the elongation conditions but readily cleaved later.Examples include: oxime resin (DeGrado and Kaiser (1980) J. Org. Chem.45, 1295-1300) chloro or bromomethyl resin, hydroxymethyl resin, andaminomethyl resin. Many of these resins are commercially available withthe desired C-terminal amino acid already incorporated.

The α-amino group of each amino acid is typically protected, e.g., by anα-amino protecting group. Any protecting group known in the art may beused. Examples of these are:

(1) acyl types such as formyl, trifluoroacetyl, phthalyl, andp-toluenesulfonyl;

(2) aromatic carbamate types such as benzyloxycarbonyl (Cbz) andsubstituted benzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxycarbonyl,and 9-fluorenyl-methyloxycarbonyl (Fmoc);

(3) aliphatic carbamate types such as tert-butyloxycarbonyl (Boc),ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl;

(4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl andadamantyloxycarbonyl;

(5) alkyl types such as triphenylmethyl and benzyl;

(6) trialkylsilane such as trimethylsilane; and

(7) thiol containing types such as phenylthiocarbonyl anddithiasuccinoyl.

Typical α-amino protecting groups are either Boc or Fmoc. Many aminoacid or amino acid mimetic derivatives suitably protected for peptidesynthesis are commercially available.

The α-amino protecting group is cleaved prior to the coupling of thenext amino acid. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane. Theresulting ammonium salt is then neutralized either prior to the couplingor in situ with basic solutions such as aqueous buffers, or tertiaryamines in dichloromethane or dimethylformamide. When the Fmoc group isused, the reagents of choice are piperidine or substituted piperidinesin dimethylformamide, but any secondary amine or aqueous basic solutionscan be used. The deprotection is carried out at a temperature between 0°C. and room temperature.

The amino acids or amino acid mimetics bearing side chainfunctionalities are typically protected during the preparation of thepeptide using any of the above-identified groups. Those skilled in theart will appreciate that the selection and use of appropriate protectinggroups for these side chain functionalities will depend upon the aminoacid or amino acid mimetic and presence of other protecting groups inthe peptide, polypeptide or peptidomimetic. The selection of such aprotecting group is important in that it must not be removed during thedeprotection and coupling of the α-amino group.

For example, when Boc is chosen for the α-amine protection the followingprotecting groups are acceptable: p-toluenesulfonyl (tosyl) moieties andnitro for arginine; benzyloxycarbonyl, substituted benzyloxycarbonyls,tosyl or trifluoroacetyl for lysine; benzyl or alkyl esters such ascyclopentyl for glutamic and aspartic acids; benzyl ethers for serineand threonine; benzyl ethers, substituted benzyl ethers or2-bromobenzyloxycarbonyl for tyrosine; p-methylbenzyl, p-methoxybenzyl,acetamidomethyl, benzyl, or tert-butylsulfonyl for cysteine; and theindole of tryptophan can either be left unprotected or protected with aformyl group.

When Fmoc is chosen for the α-amine protection usually tert-butyl basedprotecting groups are acceptable. For instance, Boc can be used forlysine, tert-butyl ether for serine, threonine and tyrosine, andtert-butyl ester for glutamic and aspartic acids.

Once the elongation of the peptide, polypeptide or peptidomimetic, orthe elongation and cyclization of a cyclic peptide or peptidomimetic iscompleted all of the protecting groups are removed. For the liquid phasesynthesis the protecting groups are removed in whatever manner asdictated by the choice of protecting groups. These procedures are wellknown to those skilled in the art.

When a solid phase synthesis is used to synthesize a cyclic peptide orpeptidomimetic, the peptide or peptidomimetic should be removed from theresin without simultaneously removing protecting groups from functionalgroups that might interfere with the cyclization process. Thus, if thepeptide or peptidomimetic is to be cyclized in solution, the cleavageconditions need to be chosen such that a free α-carboxylate and a freeα-amino group are generated without simultaneously removing otherprotecting groups. Alternatively, the peptide or peptidomimetic may beremoved from the resin by hydrazinolysis, and then coupled by the azidemethod. Another very convenient method involves the synthesis ofpeptides or peptidomimetics on an oxime resin, followed byintramolecular nucleophilic displacement from the resin, which generatesa cyclic peptide or peptidomimetic (Tetrahedron Letters, 1990, 43,6121-6124). When the oxime resin is employed, the Boc protection schemeis generally chosen. Then, a typical method for removing side chainprotecting groups generally involves treatment with anhydrous HFcontaining additives such as dimethyl sulfide, anisole, thioanisole, orp-cresol at 0° C. The cleavage of the peptide or peptidomimetic can alsobe accomplished by other acid reagents such as trifluoromethanesulfonicacid/trifluoroacetic acid mixtures.

Unusual amino acids used in this disclosure can be synthesized bystandard methods familiar to those skilled in the art (The Peptides:Analysis, Synthesis, Biology, Vol. 5, pp. 342-449, Academic Press, NewYork (1981)). N-Alkyl amino acids can be prepared using proceduresdescribed previously (Cheung et al., Can. J. Chem., 1977, 55, 906;Freidinger et al., J. Org. Chem., 1982, 48, 77).

The chelator is selected to form stable complexes with the metal ionchosen for a particular application. Chelators for diagnosticradiopharmaceuticals are selected to form stable complexes with theradioisotopes that have imagable gamma ray or positron emissions, suchas ¹¹¹In, ⁶²Cu, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ¹⁵³Gd.

Chelators for copper and gallium isotopes are selected fromdiaminedithiols, monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Thechelators are generally tetradentate with donor atoms selected fromnitrogen, oxygen and sulfur. The thiol sulfur atoms and the hydrazinesmay bear a protecting group which can be displaced either prior to usingthe reagent to synthesize a radiopharmaceutical or more often in situduring the synthesis of the radiopharmaceutical.

Exemplary thiol protecting groups include those listed in Greene andWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, NewJersey (2007). Any thiol protecting group known in the art may be used.Examples of thiol protecting groups include, but are not limited to, thefollowing: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, andtriphenylmethyl.

Chelators and chelator moieties for such metals as indium (e.g. ¹¹¹In),yttrium (e.g. ⁸⁶Y & ⁹⁰Y), and lanthanides (e.g. Eu(III), Gd(III), andDy(III)) are selected from cyclic and acyclic polyaminocarboxylates suchas DTPA, DOTA, DO3A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.Additional chelators suitable for use in the inventions are described inU.S. Pat. No. 5,362,475; U.S. Pat. No. 6,676,929; and U.S. Pat. No.7,060,250, each of which is incorporated here by reference in itsentirety. Procedures for synthesizing these chelators that are notcommercially available can be found in J. Chem. Soc. Perkin Trans.,1992, 1, 1175; Bioconjugate Chem., 1991, 2, 187; J. Nucl. Med., 1990,31, 473; U.S. Pat. No. 5,064,956; and U.S. Pat. No. 4,859,777, each ofwhich is incorporated here by reference in its entirety.

The coordination sphere of metal ion includes all the ligands or groupsbound to the metal. For a transition metal complex to be stable ittypically has a coordination number (number of donor atoms) comprised ofan integer greater than or equal to 4 and less than or equal to 8; thatis there are 4 to 8 atoms bound to the metal and it is said to have acomplete coordination sphere. For a lanthanide series or actinide seriesmetal complex, the metal typically has a coordination number (number ofdonor atoms) comprised of an integer greater than or equal to 4 and lessthan or equal to 10; that is there are 4 to 10 atoms bound to the metaland it is said to have a complete coordination sphere. The requisitecoordination number for a stable metallopharmaceutical complex isdetermined by the identity of the element, its oxidation state, and thetype of donor atoms. If the chelator does not provide all of the atomsnecessary to stabilize the metal complex by completing its coordinationsphere, the coordination sphere is completed by donor atoms from otherligands, termed ancillary or co-ligands, which can also be eitherterminal or chelating.

Ancillary ligands A_(L1) are comprised of one or more hard donor atomssuch as oxygen and amine nitrogen (sp³ hybridized). The donor atomsoccupy at least one of the sites in the coordination sphere of theradionuclide metal; the ancillary ligand A_(L1) serves as one of theligands in the ligand system. Examples of ancillary ligands A_(L1)include but are not limited to water, dioxygen ligands andfunctionalized aminocarboxylates. A large number of such ligands areavailable from commercial sources.

Ancillary dioxygen ligands include ligands that coordinate to the metalion through at least two oxygen donor atoms. Examples include but arenot limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate,lactate, tartrate, mannitol, glucarate, maltol, Kojic acid,2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzenedisulfonate, or substituted or unsubstituted 1,2- or3,4-hydroxypyridinones. (The names for the ligands in these examplesrefer to either the protonated or non-protonated forms of the ligands.)

Functionalized aminocarboxylates include ligands that have a combinationof amine nitrogen and oxygen donor atoms. Examples include but are notlimited to: iminodiacetic acid, 2,3-diaminopropionic acid,nitrilotriacetic acid, N,N′-ethylenediamine diacetic acid,N,N,N′-ethylenediamine triacetic acid, hydroxyethylethylenediaminetriacetic acid, and N,N′-ethylenediamine bis-hydroxyphenylglycine. (Thenames for the ligands in these examples refer to either the protonatedor non-protonated forms of the ligands.)

Chelators for magnetic resonance imaging contrast agents are selected toform stable complexes with paramagnetic metal ions, such as Gd(III),Dy(III), Fe(III), and Mn(II), are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

As noted above, methods for treating a patient are provided. The methodmay comprise administration of a compound or diagnostic agent describedherein to a patient and acquiring an image of a site of concentration ofthe diagnostic agent in the patient by a diagnostic imaging technique.The treatment may include the detection, imaging, and/or monitoring ofelastin-rich tissues in a patient, including elastin-rich tissueslocated within the arterial wall, uterus, lung, skin, and/or ligaments.In some cases, the treatment includes the detection, imaging, and/ormonitoring of the presence and/or amount of coronary plaque, carotidplaque, iliac/femoral plaque, aortic plaque, renal artery plaque, plaqueof any arterial vessel, aneurism, vasculitis, other diseases of thearterial wall, and/or damage or structural changes in ligaments, uterus,lungs or skin in a patient.

The rate of clearance from the blood is of particular importance forcardiac imaging procedures, since the cardiac blood pool is largecompared to the disease foci that one desires to image. For an effectivearterial wall imaging agent, the target to background ratios (diseasefoci-to-blood and disease foci-to-muscle) are typically greater or equalto about 1.5, typically greater or equal to about 2.0, and moretypically even greater. Certain pharmaceuticals of the presentdisclosure have blood clearance rates that result in less than about 5%i.d./g at 1 hour post-injection, measured in a mouse model. In oneembodiment diagnostic agents of the present disclosure have bloodclearance rates that result in less than about 2% i.d./g at 1 hourpost-injection, measured in a mouse model.

The indium, copper, gallium, and yttrium diagnostic agents of thepresent disclosure can be easily prepared by admixing a salt of aradionuclide and a reagent of the present disclosure in an aqueoussolution at temperatures from about 0° C. to about 100° C. Theseradionuclides are typically obtained as a dilute aqueous solution in amineral acid, such as hydrochloric, nitric or sulfuric acid. Theradionuclides are combined with from one to about one thousandequivalents of the reagents of the present disclosure dissolved inaqueous solution. A buffer is typically used to maintain the pH of thereaction mixture from about 3 to about 10.

The gadolinium, dysprosium, iron and manganese diagnostic agents of thepresent disclosure can be easily prepared by admixing a salt of theparamagnetic metal ion and a reagent of the present disclosure in anaqueous solution at temperatures from about 0° C. to about 100° C. Theseparamagnetic metal ions are typically obtained from commercial sourcesas their oxide, chloride or nitrate salts. The paramagnetic metal ionsare combined with from one to about one thousand equivalents of thereagents of the present disclosure dissolved in aqueous solution. Abuffer is typically used to maintain the pH of the reaction mixture fromabout 3 to about 10.

The total time of preparation will vary depending on the identity of themetal ion, the identities and amounts of the reactants and the procedureused for the preparation. The preparations may be complete, resulting ingreater than about 80% yield of the radiopharmaceutical, in about 1minute or may require more time. If higher purity metallopharmaceuticalsare needed or desired, the products can be purified by any of a numberof techniques well known to those skilled in the art such as liquidchromatography, solid phase extraction, solvent extraction, dialysis orultrafiltration.

The diagnostic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of about 1 to about 100mCi per 70 kg body weight, or typically at a dose of about 5 to about 50mCi. Imaging is performed using known procedures.

The diagnostic agents of the disclosure containing a magnetic resonanceimaging contrast component may be used in a similar manner as other MRIagents as described in U.S. Pat. No. 5,155,215; U.S. Pat. No. 5,087,440;Magn. Reson. Med., 1986, 3, 808; Radiology, 1988, 166, 835; andRadiology, 1988, 166, 693. Generally, sterile aqueous solutions of thecontrast agents are administered to a patient intravenously in dosagesranging from about 0.01 to about 1.0 mmoles per kg body weight.

For use as X-ray contrast agents, the diagnostic agents of the presentdisclosure should generally have a heavy atom concentration of about 1mM to about 5 M, typically about 0.1 M to about 2 M. Dosages,administered by intravenous injection, will typically range from about0.5 mmol/kg to 1.5 mmol/kg, typically about 0.8 mmol/kg to 1.2 mmol/kg.Imaging is performed using known techniques, typically X-ray computedtomography.

The diagnostic agents of the disclosure containing ultrasound contrastcomponents are administered by intravenous injection in an amount ofabout 10 to about 30 μL of the echogenic gas per kg body weight or byinfusion at a rate of about 3 μL/kg/min. Imaging may be performed usingknown techniques of sonography.

Other features of the disclosure will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the disclosure and are not intended to be limitingthereof. The present disclosure will now be illustrated by reference tothe following specific, non-limiting examples. Those skilled in the artof organic synthesis may be aware of still other synthetic routes to thedisclosure compounds and/or diagnostic agents. The reagents andintermediates used herein are either commercially available or preparedaccording to standard literature procedures, unless otherwise described.

This disclosure is intended to encompass compounds having formula (I)when prepared by synthetic processes or by metabolic processes includingthose occurring in the human or animal body (in vivo) or processesoccurring in vitro. For example, compounds of the present disclosurewhere A is a peptide consisting of a D-amino acid residue and a secondD-amino acid may be generated by cleavage of a larger sequence (e.g., apeptide consisting of 3 amino acids and a D-amino acid residue) eithersynthetically or in vivo.

Example 12-{[2-({[N-({4-[((2R)-2-amino-4-phenylbutanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation ofN-(1-(N-hydroxycarbamoyl)(1R)-3-phenylpropyl)(tert-butoxy)-carboxamide

A solution of Boc-DHfe-OH (1.40 g, 5.00 mmol) in 4:1 CH₂Cl₂/MeOH (25.0mL) was treated with (trimethylsilyl)diazomethane (6.00 mmol; 3.00 mL ofa 2.0 M solution in Et₂O) dropwise over 0.25 h at 22° C. CAUTION:vigorous gas evolution. The resulting yellow solution was stirred anadditional 0.25 h to ensure complete methylation (R_(f)=0.7 in 1:1EtOAc/hexanes). Excess (trimethylsilyl)diazomethane was consumed by thedropwise addition of glacial AcOH, then all volatiles removed in vacuo.The crude ester was redissolved in MeOH (25.0 mL), cooled to 0° C. andtreated with a previously prepared suspension of H₂NOH.HCl (1.04 g, 15.0mmol) and KOH (1.68 g, 30.0 mmol) in MeOH (25.0 mL); a large borecannula needle was required for the transfer. The resulting suspensionthen warmed slowly to 22° C. over 3.5 h as the ice bath melted; thesuspension stirred at 22° C. for 0.75 h of the time interval. Thesuspension was acidified with conc. HCl to pH 4-5 then all volatilesremoved in vacuo. The solids were triturated with several portions ofhot EtOAc (5×10 mL) and removed by filtration through a scintered glassfunnel of medium porosity. The combined filtrates were collected andconcentrated in vacuo to an off-white powder (R_(f)=0.7 in 9:1CH₂Cl₂/MeOH). Purification through recrystallization from hot EtOAc (150mL) afforded a white microcrystalline solid (0.893 g, 3.03 mmol; 60.6%).Mp 165.5-166.0° C. ¹H NMR (DMSO-d₆, 300 MHz): δ 10.5 (1H, brs), 8.79(1H, brs), 7.30-7.25 (2H, m), 7.19-7.14 (3H, m), 6.96 (1H, brd, J=8.1Hz), 3.82 (1H, dt, J=7.5, 7.5 Hz), 2.66-2.45 (2H, m), 1.87-1.74 (2H, m),1.39 (9H, s). ¹³C NMR (DMSO-d₆, 75 MHz): δ 168.8, 155.2, 141.3, 128.3,128.2, 125.7, 77.9, 51.8, 33.8, 31.6, 28.2. HRMS calcd for C₁₅H₂₂N₂O₄(M+Na): 317.1472. Found: 317.1466. The optical purity of the product wasestablished by chiral GLC analysis (99.9% D-homophenylalanine).

Part B—Preparation ofN-{[4-(hydroxymethyl)phenyl]methyl}prop-2-enyloxycarboxamide

A suspension of methyl 4-(aminomethyl)benzoate hydrochloride (1.01 g,5.00 mmol) in THF (50.0 mL) was treated with i-Pr₂NEt (2.09 mL, 12.0mmol) then cooled to 0° C. Allyl chloroformate (638 μL, 6.00 mmol) wasthen added over 10 min and the resulting suspension stirred 50 min at 0°C. The reaction mixture was diluted with H₂O (50 mL), the layersseparated and the aqueous layer washed with Et₂O (3×50 mL). The combinedTHF and Et₂O solutions were dried over MgSO₄, filtered and concentratedin vacuo to a white solid (R_(f)=0.5 in 1:1 hexanes/EtOAc) which wasused without further purification in the subsequent reduction step.

The crude ester (5.00 mmol theoretical) was dissolved in dry THF (20.0mL), cooled to 0° C. and treated with LiAlH₄ (5.00 mmol; 5.00 mL of a 1M solution in THF) dropwise over 0.25 h using a syringe pump. Theresulting solution was stirred 0.25 h at 0° C. to ensure completereduction. Excess LiAlH₄ was consumed by the careful addition of H₂O(200 μL). The resulting white suspension was successively treated with15% aqueous NaOH (200 μL) and H₂O (600 μL) then stirred for 0.25 h to afine white slurry. The resulting mixture was filtered through a pad ofCelite and concentrated in vacuo. The crude oil was purified bychromatography on silica (40×185 mm) using 1:1 hexanes/EtOAc(R_(f)=0.3). The main product eluted between 430-680 mL, was collectedand concentrated to afford a white crystalline solid (0.923 g, 4.17mmol; 83.4% over two steps). Mp 80.0-81.0° C. ¹H NMR (CDCl₃, 300 MHz): δ7.32 (2H, AB, J_(AB)=8.2 Hz), 7.26 (2H, AB, J_(AB)=8.2 Hz), 5.91 (1H,ddt, J=17.2, 10.4, 5.6 Hz), 5.30 (1H, dq, J=17.2, 1.4 Hz), 5.20 (1H, dq,J=10.5, 1.3 Hz), 4.65 (2H, s), 4.58 (2H, brdt, J=5.6, 1.3 Hz), 4.34 (2H,brd, J=5.7 Hz), 1.85 (1H, s). ¹³C NMR (CDCl₃, 75 MHz): δ 156.3, 140.3,137.8, 132.8, 127.7, 127.3, 117.7, 65.7, 64.9, 44.8. HRMS calcd forC₁₂H₁₅NO₃ (M+H): 222.1125. Found: 222.1124.

Part C—Preparation ofN-{[4-(bromomethyl)phenyl]methyl}prop-2-enyloxycarboxamide

A solution of the product of Part 1B (0.664 g, 3.00 mmol) and CBr₄ (1.19g, 3.60 mmol) in dry CH₂Cl₂ (30.0 mL) was cooled to 0° C. and treatedwith PPh₃ (0.905 g, 3.45 mmol) portion-wise over 5 min. After 10 min at0° C., the solution was warmed to 22° C., stirred 20 min thenconcentrated in vacuo. The crude residue was purified by chromatographyon silica (25×170 mm) using 3:2 hexanes/EtOAc (R_(f)=0.6 in 1:1hexanes/EtOAc). The main product eluted between 95-185 mL, was collectedand concentrated to afford a white crystalline solid (0.738 g, 2.60mmol; 86.6%). Mp 80.0-82.0° C. ¹H NMR (CDCl₃, 300 MHz): δ 7.36 (2H,AA′BB′. J_(AB)=8.2 Hz, J_(AA′)=1.9 Hz), 7.26 (2H, AB, J_(AB)=8.1 Hz),5.92 (1H, ddt, J=17.2, 10.4, 5.6 Hz), 5.30 (1H, brd, J=17.0 Hz), 5.21(1H, dq, J=10.4, 1.3 Hz), 4.59 (2H, brdt, J=5.6, 1.2 Hz), 4.47 (2H, s),4.36 (2H, d, J=6.1 Hz). ¹³C NMR (CDCl₃, 75 MHz): δ 156.2, 138.9, 137.1,132.8, 129.4, 127.9, 117.8, 65.8, 44.7, 33.1. HRMS calcd forC₁₂H₁₄BrNO₂(M+H): 284.0281. Found: 284.0280.

Part D—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonyl-amino]-4-phenylbutanamide,trifluoroacetic acid salt

A solution of the product of Part 1A (0.662 g, 2.25 mmol) in dry DMF(9.00 mL) was treated with K₂CO₃ (0.373 g, 2.70 mmol) and cooled to 0°C. Part 1C (0.256 g, 0.900 mmol) was then added in one portion and theresulting suspension warmed slowly to 22° C. overnight as the ice bathmelted. After 13 h total, the reaction mixture was partitioned betweenEtOAc (150 mL) and H₂O (50 mL) with transfer to a separatory funnel. Thelayers were separated and the EtOAc layer washed with saturated aqueousNaCl (3×50 mL) then dried over MgSO₄, filtered and concentrated in vacuoto a white powder that was used without further purification in thesubsequent deprotection step (R_(f)=0.4 in 1:1 hexanes/EtOAc).

The crude hydroxamate ester (0.900 mmol theoretical) was dissolved in2:1 MeCN/H₂O (9.00 mL) and successively treated with 51.2 mg TPPTS (90.0μmol; 10 mol %), Et₂NH (233 μL, 2.25 mmol) and 10.1 mg Pd(OAc)₂ (45.0μmol; 5 mol %) at 22° C. Complete deprotection was observed within 0.5h. The amber solution was filtered through a 0.45 μm Acrodisk thendirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 0-40% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 32 min was collected andlyophilized to a white powder (158 mg, 0.300 mmol; 33.3%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.25 (1H, brs), 8.20 (3H, brs), 7.44 (4H, brs),7.27 (2H, dd, J=7.6, 7.6 Hz), 7.17 (1H, t, J=7.3 Hz), 7.15 (2H, d, J=7.3Hz), 7.08 (1H, brd, J=7.6 Hz), 4.78 (2H, brs), 4.02 (2H, brs), 3.76 (1H,dt, J=7.3, 7.1 Hz), 2.60-2.55 (1H, m), 2.50-2.45 (1H, m), 1.81-1.77 (2H,m), 1.39 (9H, s). ¹³C NMR (DMSO-d₆, 151 MHz): δ 169.0, 157.8 (q, J=31.1Hz), 155.2, 141.2, 136.3, 133.8, 128.8, 128.7, 128.2, 125.8, 117.2, (q,J=300 Hz), 78.1, 76.3, 51.9, 42.0, 33.5, 31.5, 28.2. HRMS calcd forC₂₃H₃₁N₃O₄ (M+H): 414.2387. Found: 414.2392.

Part E—Preparation of2-{[2-({[N-({4-[((2R)-2-amino-4-phenylbutanoylaminooxy)methyl]-phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl]-(carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (24.2 mg, 39.2 μmol; for leading references on the synthesis andcharacterization of DTPA and related analogs, see: a) Williams, M. A.;Rapoport, H. J. Org. Chem. 1993, 58, 1151. b) Anelli, P. L.; Fedeli, F.;Gazzotti, O.; Lattuada, L.; Lux, G.; Rebasti, F. Bioconjugate Chem.1999, 10, 137.) in dry DMF (3.27 mL) was successively treated with HOBt(6.0 mg, 39 μmol), i-Pr₂NEt (14 μL, 78 μmol) and HBTU (14.9 mg, 39.2μmol) at 22° C. After 0.25 h, the solution was transferred using acannula to the product of Part 1D (15.0 mg, 32.7 μmol) and the resultingsolution stirred 0.25 h. To complete conversion, the solution wasfurther treated with HBTU (7.43 mg, 19.6 μmol) and i-Pr₂NEt (28.0 μL,161 μmol), stirred 0.25 h, then partitioned between EtOAc and 0.1 Mcitric acid (30 mL each) with transfer to a separatory funnel. Thelayers separated and the aqueous layer washed with EtOAc (2×30 mL). Thecombined EtOAc layers were successively washed with 0.1 M citric acidand saturated aqueous solutions of NaHCO₃ and NaCl (3×30 mL each) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step (R_(f)=0.4 in 9:1 CH₂Cl₂/MeOH).

The protected conjugate (32.7 μmol theoretical) was dissolved in dioxane(650 μL) then successively treated with H₂O (3 μL) and HCl (2.60 mmol;0.650 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred and monitored over 4 h during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O containing 0.1% TFA (8.50 mL) then directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 0-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 25 min was collected and lyophilized toa white powder (22.8 mg, 22.1 μmol; 67.6%). ¹H NMR (DMSO-d₆, 300 MHz): δ11.77 (1H, brs), 8.95 (1H, brt, J=4.9 Hz), 8.35 (3H, brs), 7.40 (2H, AB,J_(AB)=8.0 Hz), 7.32-7.27 (4H, m), 7.20 (1H, dd, J=7.4, 7.4 Hz), 7.13(2H, AB, J_(AB)=7.2 Hz), 4.84 (2H, AB, J=11.6 Hz), 4.34 (2H, brd, J=5.6Hz), 4.25 (2H, s), 3.64 (1H, brs), 3.50 (8H, s), 3.38 (4H, brt, J=5.6Hz), 3.05 (4H, brt, J=5.7 Hz), 2.55-2.50 (2H, m), 1.97-1.90 (2H, m). ¹³CNMR (DMSO-d₆, 151 MHz): δ 172.7, 165.3, 164.8, 158.0 (q, J=32.4 Hz),140.2, 138.7, 134.3, 129.0, 128.5, 128.1, 127.3, 126.2, 116.8 (q, J=298Hz), 76.9, 54.3, 53.9, 52.2, 50.3, 48.6, 42.1, 32.8, 30.3. HRMS calcdfor C₃₂H₄₄N₆O₁₁ (M+Na): 711.2960. Found: 711.2964. The optical purity ofthe product was established by chiral GLC analysis (99.8%D-homophenylalanine).

Example 22-(7-{[N-({4-[((2R)-2-amino-4-phenylbutanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

A solution of2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}-cyclododecyl)aceticacid (109 mg, 0.190 mmol) in dry DMF (10.0 mL) was successively treatedwith HOBt (29.0 mg, 0.190 mmol), HBTU (71.9 mg, 0.190 mmol) and i-Pr₂NEt(40.8 μL, 0.234 mmol) at 22° C. After 0.25 h, the solution was treatedwith the product of Part 1D (0.158 mmol; 5.80 mL of a 0.027 M solutionin DMF) and the resulting solution stirred 3 h. To complete conversionthe solution was further treated with 30 mol % of the active ester,stirred 0.25 h, then diluted with EtOAc (75 mL) with transfer to aseparatory funnel. The EtOAc solution was washed with 0.1 M citric acid(3×75 mL), followed by saturated aqueous solutions of NaHCO₃ and NaCl(3×75 mL each), then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (0.158 mmol theoretical) was dissolved indioxane (3.16 mL) then successively treated with H₂O (15 μL) and HCl(12.6 mmol; 3.16 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 16 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O (8.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from0-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 25 min was collected and lyophilized to a whitepowder (43.0 mg, 41.3 μmol; 26.1%). ¹H NMR (DMSO-d₆, 600 MHz): δ 9.04(1H, brt, J=6.0 Hz), 7.46 (2H, AB, J_(AB)=8.0 Hz), 7.38 (2H, AB,J_(AB)=8.0 Hz), 7.28-7.25 (3H, m), 7.20-7.16 (3H, m), 5.01 (2H, AB,J_(AB)=11.6 Hz), 4.47 (2H, brd, J=5.7 Hz), 4.13 (1H, t, J=6.6 Hz), 3.86(4H, s), 3.85 (2H, s), 3.73 (2H, s), 3.16 (10H, brs), 3.08 (2H, brs),2.81-2.76 (2H, m), 2.30-2.21 (2H, m). ¹³C NMR (DMSO-d₆, 151 MHz): δ171.5, 165.3, 157.8 (q, J=31.4 Hz), 140.2, 138.8, 134.3, 128.9, 128.5,128.0, 127.4, 126.2, 117.1 (q, J=299 Hz), 76.9, 54.8, 54.0, 53.1, 50.6,50.4, 50.2, 48.8, 42.0, 32.8, 30.3. HRMS calcd for C₃₄H₉N₇O₉ (M+H):700.3665. Found: 700.3659.

Example 32-{[2-({[N-({4-[((2S)-2-amino-4-phenylbutanoylaminooxy)methyl]-phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl]-(carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation ofN-(1-(N-hydroxycarbamoyl)(1S)-3-phenylpropyl)(tert-butoxy)-carboxamide

A suspension of H-Hfe-OH (1.79 g, 10.0 mmol) in 2:1 THF/H₂O (50.0 mL)was treated with Na₂CO₃ (2.54 g, 24.0 mmol) followed by Boc₂O (2.62 g,12.0 mmol) in one portion at 22° C. After 1 h the heavy suspension wasacidified to pH 3-4 using 0.1 M HCl, and the resulting homogeneoussolution transferred to a separatory funnel and washed with EtOAc (4×50mL). The combined EtOAc washes were dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil that was used without furtherpurification in subsequent reactions.

A solution of crude Boc-Hfe-OH (10.0 mmol theoretical) in 4:1CH₂Cl₂/MeOH (50.0 mL) was treated with (trimethylsilyl)diazomethane(12.0 mmol; 6.00 mL of a 2.0 M solution in Et₂O) dropwise over 0.25 h at22° C. CAUTION: vigorous gas evolution. The resulting yellow solutionwas stirred an additional 0.25 h to ensure complete methylation. Excess(trimethylsilyl)diazomethane was consumed by the dropwise addition ofglacial AcOH, then all volatiles removed in vacuo. The crude ester wasredissolved in MeOH (50.0 mL), cooled to 0° C. and treated with apreviously prepared suspension of H₂NOH.HCl (2.08 g, 30.0 mmol) and KOH(3.37 g, 60.0 mmol) in MeOH (50.0 mL); a large bore cannula needle wasrequired for the transfer. The resulting suspension then warmed slowlyto 22° C. overnight as the ice bath melted. After 14 h, the suspensionwas acidified with conc. HCl to pH 4-5 then all volatiles removed invacuo. The solids were triturated with several portions of hot EtOAc(5×10 mL) and removed by filtration through a scintered glass funnel ofmedium porosity. The combined filtrates were collected and concentratedin vacuo to an off-white powder. Purification through recrystallizationfrom hot EtOAc (200 mL) afforded a white microcrystalline solid (1.47 g,4.99 mmol, 49.9%). The spectral data obtained for this material are inaccord with that described for the product of Part 1A.

Part B—Preparation of(2S)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonyl-amino]-4-phenylbutanamide,formic acid salt

A solution of K₂CO₃ (0.207 g, 1.50 mmol) in H₂O (3.00 mL) was dilutedwith absolute EtOH (7.00 mL) then treated with the product of Part 3A(0.442 g, 1.50 mmol) in one portion at 22° C. Upon complete dissolution(10-15 min), the product of Part 1C (0.284 g, 1.00 mmol) was added inone portion and the resulting suspension stirred vigorously; a rapidstirring rate is required to ensure complete dissolution of the bromide.Within 25 min the solution turned cloudy and a heavy white precipitateformed; the reaction was complete at 1 h. The resulting suspension wasthen diluted with H₂O (40 mL) and the solids collected on a scinteredglass funnel of medium porosity. The solids were further washed with H₂Oand Et₂O (5×20 mL each) then dried in vacuo to a white powder that wasused without further purification in the subsequent deprotection step.

The hydroxamate ester (0.337 g, 0.677 mmol) was dissolved in 2:1MeCN/H₂O (6.77 mL) and successively treated with 15.4 mg TPPTS (27.1μmol; 4 mol %), Et₂NH (175 μL, 1.69 mmol) and 3.0 mg Pd(OAc)₂ (13.5μmol; 2 mol %) at 22° C. Complete deprotection was observed within 1 h.The amber solution was diluted to 14 mL with H₂O containing 0.1% HCO₂H,then filtered through a 0.45 μm Acrodisk and purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from10-50% MeCN containing 0.1% HCO₂H and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 17 min was collected and lyophilized to a whitepowder (0.229 g, 0.498 mmol; 49.8%). The spectral data obtained for thismaterial are in accord with that described for the product of Part 1B.

Part C—Preparation of2-{[2-({[N-({4-[((2S)-2-amino-4-phenylbutanoylaminooxy)methyl]-phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl]-(carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (0.278 g, 0.450 mmol) in dry DMF (3.00 mL) was successively treatedwith HOBt (68.9 mg, 0.450 mmol), i-Pr₂NEt (131 μL, 0.750 mmol) and HBTU(0.171 g, 0.450 mmol) at 22° C. After 0.25 h, the solution wastransferred to the product of Part 3B (0.138 g, 0.300 mmol) using acannula. The resulting solution was stirred 0.5 h then partitionedbetween EtOAc and 0.1 M citric acid (50 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×50 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×50 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (0.300 mmol theoretical) was dissolved indioxane (3.00 mL) then successively treated with H₂O (27 μL) and HCl(12.0 mmol; 3.00 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 15 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed in vacuo and the white solid residue redissolvedin H₂O containing 0.1% TFA (8.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from0-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 25 min was collected and lyophilized to a whitepowder (0.181 g, 0.176 mmol; 58.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.79(1H, brs), 8.96 (1H, brt, J=5.9 Hz), 8.37 (3H, brs), 7.39 (2H, AB,J_(AB)=8.1 Hz), 7.32-7.29 (4H, m), 7.20 (1H, brdd, J=7.3, 7.3 Hz), 7.13(2H, AB, J_(AB)=7.1 Hz), 4.84 (2H, AB, J_(AB)=11.8 Hz), 4.34 (2H, brd,J=5.8 Hz), 4.25 (2H, brs), 3.65 (1H, brs), 3.50 (8H, s), 3.38 (4H, brt,J=5.8 Hz), 3.05 (4H, brt, J=5.9 Hz), 2.54-2.50 (2H, m), 1.96-1.91 (2H,m). ¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 165.3, 164.8, 158.1 (q, J=32.2Hz), 140.2, 138.7, 134.3, 128.9, 128.5, 128.1, 127.3, 126.2, 116.9 (q,J=299 Hz), 76.9, 54.3, 53.9, 52.2, 50.2, 48.7, 42.1, 32.8, 30.3. HRMScalcd for C₃₂H₄N₆O₁₁ (M+H): 689.3141. Found: 689.3147. The opticalpurity of the product was established by chiral GLC analysis (99.0%L-homophenylalanine).

Example 42-({2-[({N-[6-((2R)-2-amino-4-methylpentanoylaminooxy)hexyl]carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)amino)aceticacid, trifluoroacetic acid salt

Part A—Preparation of 6-(Prop-2-enyloxycarbonylamino)hexylmethylsulfonate

A solution of N-(6-hydroxyhexyl)prop-2-enyloxycarboxamide (2.55 g, 12.7mmol; Charreyre, M. T.; Boullanger, P.; Pichot, C.; Delair, T.;Mandrand, B.; Llauro, M. F. Mak. Chem. 1993, 194(1), 117-35.) in dryCH₂Cl₂ (30.0 mL) was treated with Et₃N (4.06 mL, 29.1 mmol) then cooledto 0° C. To this solution was transferred MsCl (15.2 mmol; 20.0 mL of a0.76 M solution in CH₂Cl₂) using a cannula; full conversion coincidedwith completion of the transfer. The resulting solution was warmed to22° C., then treated with 2 M NH₄Cl (50 mL) and transferred to aseparatory funnel. The layers separated and the aqueous layer washedwith CH₂Cl₂ (3×50 mL). The combined washes were washed with 20% aqueousNaCl then dried over MgSO₄, filtered and concentrated in vacuo to a paleyellow oil (3.2 g) that was used without further purification in thesubsequent alkylation step. ¹H NMR (CDCl₃, 300 MHz): δ 5.90 (1H, ddt,J=17.2, 10.4, 5.6 Hz), 5.29 (1H, dq, J=17.2, 1.6 Hz), 5.19 (1H, dq,J=10.4, 1.3 Hz), 4.71 (1H, brs), 4.54 (2H, brd, J=5.5 Hz), 4.20 (2H, t,J=6.5 Hz), 3.17 (2H, brs), 2.98 (3H, s), 1.79-1.69 (2H, m), 1.55-1.29(6H, m).

Part B—Preparation ofN-{6-[(tert-butoxy)carbonylaminooxy]hexyl}prop-2-enyloxycarboxamide

A solution of N-Boc hydroxylamine (2.37 g, 17.8 mmol) in anhydrous Et₂O(5.00 mL) was treated with DBU (2.85 mL, 19.1 mmol) then cooled to 0° C.To this mixture was transferred the product of Part 4A (12.7 mmol; 5.00mL of a 2.53 M solution in Et₂O) by means of a cannula. The resultingsolution then warmed slowly to 22° C. overnight as the ice bath melted.After 17 h the Et₂O was removed under a stream of N₂, and the resultingthick oil stirred 16 h to ensure complete conversion. After this time,the solution was diluted with Et₂O (20 mL), transferred to a separatoryfunnel then successively washed with 2 M NH₄Cl (30 mL) and 20% aqueousNaCl (2×30 mL). The resulting Et₂O solution was dried over MgSO₄,filtered and concentrated in vacuo to a pale yellow oil that waspurified by chromatography on silica (3:1 hexanes/EtOAc; R_(f)=0.5 in2:1 hexanes/EtOAc) to afford a colorless oil (2.61 g, 8.25 mmol; 65.1%).¹H NMR (CDCl₃, 600 MHz): δ 7.14 (1H, brs), 5.91 (1H, ddt, J=17.2, 10.5,5.7 Hz), 5.29 (1H, dq, J=17.2, 1.6 Hz), 5.19 (1H, dq, J=10.5, 1.4 Hz),4.77 (1H, brs), 4.55 (2H, brd, J=5.0 Hz), 3.83 (2H, t, J=6.5 Hz), 3.17(2H, dt, J=6.7, 6.4 Hz), 1.63-1.59 (2H, m), 1.52-1.47 (2H, m), 1.47 (9H,s), 1.42-1.31 (4H, m).

Part C—Preparation of N-[6-(aminooxy)hexyl]prop-2-enyloxycarboxamide,hydrochloric acid salt

The product of Part 4B (2.61 g, 8.25 mmol) was treated with HCl (16.0mmol; 8.00 mL of a 2 M solution in Et₂O), and the resulting solutionstirred 5 h at 22° C. The heavy white precipitate that formed wascollected on a scintered glass funnel, then washed with Et₂O (3×8 mL)and dried to constant weight in vacuo (1.02 g, 4.04 mmol; 48.9%). Theresulting material required no additional purification. ¹H NMR (DMSO-d₆,600 MHz): δ 10.95 (3H, brs), 7.15 (1H, brt, J=5.5 Hz), 5.89 (1H, ddt,J=17.2, 10.5, 5.2 Hz), 5.25 (1H, dq, J=17.2, 1.8 Hz), 5.15 (1H, dq,J=10.5, 1.6 Hz), 4.43 (2H, brd, J=5.5 Hz), 3.98 (2H, t, J=6.5 Hz),2.97-2.93 (2H, m), 1.57-1.53 (2H, m), 1.38 (2H, tt, J=7.1, 7.1 Hz),1.31-1.22 (4H, m). ¹³C NMR (DMSO-d₆, 151 MHz): δ 155.8, 133.8, 116.7,73.9, 64.0, 40.0, 29.2, 27.0, 25.7, 24.7. HRMS calcd for C₁₀H₂₀N₂O₃(M+Na): 239.1366. Found: 239.1363.

Part D—Preparation of(2R)—N-(6-aminohexyloxy)-2-[(tert-butoxy)carbonylamino]-4-methylpentanamide,trifluoroacetic acid salt

A solution of Boc-DLeu-OH (0.231 g, 1.00 mmol) in MeCN (4.00 mL) wassuccessively treated with HOBt (0.153 g, 1.00 mmol), i-Pr₂NEt (174 μL,1.00 mmol) and HBTU (0.379 g, 1.00 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 4C (0.210 g, 0.831 mmol)in one portion. The resulting solution was stirred 1 h then partitionedbetween CH₂Cl₂ and 0.1 M citric acid (50 mL each) with transfer to aseparatory funnel. The layers separated and the CH₂Cl₂ solutionsuccessively washed with 0.1 M citric acid (2×50 mL) and saturatedaqueous solutions of NaHCO₃ (3×50 mL) and NaCl (50 mL) then dried overMgSO₄, filtered and concentrated in vacuo to a colorless oil which wasused without further purification in the subsequent deprotection step.

The crude hydroxamate ester (0.831 mmol theoretical) was dissolved in2:1 MeCN/H₂O (3.00 mL) and successively treated with 18.9 mg TPPTS (33.2μmol; 4 mol %), Et₂NH (216 μL, 2.09 mmol) and 3.7 mg Pd(OAc)₂ (16.5μmol; 2 mol %) at 22° C. Complete deprotection was observed within 0.5h. The amber solution was filtered through a 0.45 μm Acrodisk thendirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 0-40% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 34 min was collected andlyophilized to a white powder (0.190 g, 0.413 mmol; 49.8%).

Part E—Preparation of2-({2-[({N-[6-((2R)-2-amino-4-methylpentanoylaminooxy)-hexyl]carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)-amino)aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (74.0 mg, 0.120 mmol) in dry DMF (1.00 mL) was successively treatedwith HOBt (18.4 mg, 0.120 mmol), i-Pr₂NEt (35 μL, 0.20 mmol) and HBTU(45.5 mg, 0.120 mmol) at 22° C. After 0.25 h, the solution was treatedwith the product of Part 4D (46.0 mg, 0.100 mmol) in one portion. Theresulting solution was stirred 0.5 h then diluted with EtOAc (50 mL),washed with 0.1 M citric acid (3×30 mL), 0.1 M NaOH (3×30 mL) andsaturated aqueous and NaCl (30 mL) then dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil which was used without furtherpurification in the subsequent deprotection step.

The protected conjugate (0.100 mmol theoretical) was dissolved indioxane (0.500 mL) then successively treated with H₂O (2 μL) and HCl(2.00 mmol; 0.500 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 15 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residuedirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 0-40% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 20 min was collected andlyophilized to a white powder (52.0 mg, 0.054 mmol; 54.0%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.68 (1H, brs), 8.43 (1H, brt, J=5.5 Hz), 8.26(2H, brs), 4.13 (2H, s), 3.79 (2H, t, J=6.6 Hz), 3.53 (1H, brs), 3.49(8H, s), 3.34 (4H, brt, J=5.5 Hz), 3.11 (2H, td, J=6.9, 5.7 Hz), 3.03(4H, brt, J=5.9 Hz), 1.60-1.50 (5H, m), 1.43 (2H, tt, J=7.2, 7.2 Hz),1.36-1.26 (4H, m), 0.89 (3H, d, J=6.3 Hz), 0.87 (3H, d, J=6.0 Hz). ¹³CNMR (DMSO-d₆, 151 MHz): δ 172.7, 165.4, 164.4, 157.9 (q, J=31.8 Hz),117.1 (q, J=300 Hz), 75.3, 54.3, 53.9, 52.1, 48.9, 48.6, 40.0, 38.7,28.7, 27.4, 26.1, 24.9, 23.7, 22.2, 22.0. HRMS calcd for C₂₆H₄₈N₆O₁₁(M+H): 621.3454. Found: 621.3462.

Example 52-[(2-{[(N-{6-[(2R)-2-amino-3-(4-phenylphenyl)propanoylaminooxy]hexyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-(6-aminohexyloxy)-2-[(tert-butoxy)carbonylamino]-3-(4-phenylphenyl)propanamide,trifluoroacetic acid salt

A solution of Boc-DBip-OH (0.231 g, 1.00 mmol) in MeCN (4.00 mL) wassuccessively treated with HOBt (0.153 g, 1.00 mmol), i-Pr₂NEt (174 μL,1.00 mmol) and HBTU (0.379 g, 1.00 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 4C (0.210 g, 0.831 mmol)in one portion. The resulting solution was stirred 1 h then partitionedbetween EtOAc and 0.1 M citric acid (50 mL each) with transfer to aseparatory funnel. The layers separated and the EtOAc solutionsuccessively washed with 0.1 M citric acid (2×50 mL) and saturatedaqueous solutions of NaHCO₃ (3×50 mL) and NaCl (50 mL) then dried overMgSO₄, filtered and concentrated in vacuo to a white solid which wasused without further purification in the subsequent deprotection step.

The crude hydroxamate ester (0.831 mmol theoretical) was dissolved in2:1 MeCN/H₂O (3.00 mL) and successively treated with 18.9 mg TPPTS (33.2μmol; 4 mol %), Et₂NH (216 μL, 2.09 mmol) and 3.7 mg Pd(OAc)₂ (16.5μmol; 2 mol %) at 22° C. Complete deprotection was observed within 0.5h. The amber solution was filtered through a 0.45 μm Acrodisk thendirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 10-50% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 35 min was collected andlyophilized to a white powder (0.140 g, 0.246 mmol; 29.6%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.06 (1H, brs), 7.70-7.60 (4H, m), 7.56 (2H, AB,J_(AB)=8.0 Hz), 7.44 (2H, dd, J=8.0, 7.4 Hz), 7.33 (1H, brt, J=7.4 Hz),7.31 (2H, AB, J_(AB)=8.0 Hz), 7.06 (1H, brd, J=8.2 Hz), 4.02-3.99 (1H,m), 3.68-3.59 (2H, m), 2.88 (2H, ABX, J_(AB)=13.5 Hz, J_(AX)=6.1 Hz,J_(BX)=9.3 Hz), 2.73 (2H, brs), 1.52-1.42 (4H, m), 1.31 (9H, s),1.30-1.25 (4H, m).

Part B—Preparation of2-[(2-{[(N-{6-[(2R)-2-amino-3-(4-phenylphenyl)propanoylaminooxy]-hexyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}ethyl)(carboxymethyl)-amino]aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (74.0 mg, 0.120 mmol) in dry DMF (1.00 mL) was successively treatedwith HOBt (18.4 mg, 0.120 mmol), i-Pr₂NEt (35 μL, 0.20 mmol) and HBTU(45.5 mg, 0.120 mmol) at 22° C. After 0.25 h, the solution was treatedwith the product of Part 5A (57.0 mg, 0.100 mmol) in one portion. Theresulting solution was stirred 0.5 h then diluted with EtOAc (50 mL),washed with 0.1 M citric acid (3×30 mL), 0.1 M NaOH (3×30 mL) andsaturated aqueous and NaCl (30 mL) then dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil which was used without furtherpurification in the subsequent deprotection step.

The protected conjugate (0.100 mmol theoretical) was dissolved indioxane (0.500 mL) then successively treated with H₂O (2 μL) and HCl(2.00 mmol; 0.500 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 15 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residuedirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 10-40% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 10 min was collected andlyophilized to a white powder (35.0 mg, 32.6 μmol; 32.6%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.47 (1H, brs), 8.43 (2H, brs), 8.40 (1H, brd,J=5.5 Hz), 7.64-7.63 (4H, m), 7.47-7.44 (2H, m), 7.37-7.34 (1H, m), 7.30(2H, AB, J_(AB)=7.8 Hz), 4.13 (2H, s), 3.79 (1H, brs), 3.63 (1H, ABX,J_(AB)=9.7 Hz, J_(AX)=6.8 Hz), 3.53 (1H, ABX, J_(AB)=9.7 Hz, J_(BX)=6.6Hz), 3.49 (8H, s), 3.45 (4H, brt, J=5.8 Hz), 3.09-2.99 (4H, m), 3.03(4H, brt, J=6.0 Hz), 1.37-1.32 (4H, m), 1.23-1.16 (4H, m). ¹³C NMR(DMSO-d₆, 151 MHz): δ 172.7, 164.3, 164.2, 157.9 (q, J=31.8 Hz), 139.6,139.0, 133.9, 130.0, 128.9, 127.4, 126.7, 126.4, 116.9 (q, J=299 Hz),75.3, 54.3, 53.8, 52.1, 51.5, 48.6, 38.7, 36.5, 28.6, 27.3, 26.0, 24.8.HRMS calcd for C₃₅H₅₀N₆O₁₁ (M+H): 731.3610. Found: 731.3612.

Example 62-({2-[({N-[6-((2R)-2-amino-3-cyclohexylpropanoylaminooxy)hexyl]carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)amino)aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-(6-aminohexyloxy)-2-[(tert-butoxy)carbonylamino]-3-cyclohexylpropanamide,trifluoroacetic acid salt

A solution of Boc-DCha-OH (0.163 g, 0.360 mmol) in CH₂Cl₂ (3.00 mL) wassuccessively treated with HOBt (55.1 mg, 0.360 mmol), i-Pr₂NEt (125 μL,0.720 mmol) and HBTU (0.137 g, 0.360 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 4C (75.8 mg, 0.300 mmol)in one portion. The resulting solution was stirred 1 h then allvolatiles removed in vacuo. The crude hydroxamate ester was redissolvedin 2:1 MeCN/H₂O (3.00 mL) and successively treated with 17.1 mg TPPTS(30.0 μmol; 10 mol %), Et₂NH (78 μL, 0.75 mmol) and 3.4 mg Pd(OAc)₂ (15μmol; 5 mol %) at 22° C. Complete deprotection was observed within 1 h.The resulting yellow solution was diluted with H₂O containing 0.1% TFA(5.00 mL) then filtered through a 0.45 μm Acrodisk and directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 10-50% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 38 min was collected and lyophilized toa white powder (77.7 mg, 0.156 mmol; 51.8%). A small amount of TPPTS canbe detected in the ¹H NMR spectrum. ¹H NMR (DMSO-d₆, 600 MHz): δ 11.04(1H, brs), 7.71 (3H, brs), 6.84 (1H, brd, J=8.1 Hz), 3.83-3.80 (1H, m),3.73-3.67 (2H, m), 2.79-2.73 (2H, m), 1.87-1.45 (9H, m), 1.42-1.27 (5H,m), 1.36 (9H, s), 1.25-1.07 (4H, m), 0.87-0.78 (2H, m). ¹³C NMR(DMSO-d₆, 151 MHz): δ 169.1, 158.0 (q, J=31.8 Hz), 155.1, 117.0 (q,J=300 Hz), 77.9, 74.7, 49.7, 38.7, 33.5, 32.8, 32.0, 28.1, 27.4, 26.8,26.0, 25.8, 25.6, 25.5, 24.8. HRMS calcd for C₂₀H₃₉N₃O₄ (M+H): 386.3013.Found: 386.3016.

Part B—Preparation of2-({2-[({N-[6-((2R)-2-amino-3-cyclohexylpropanoylaminooxy)hexyl]-carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)-amino)aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (18.5 mg, 30.0 μmol), HOBt (4.6 mg, 30.0 μmol) and the product ofPart 6A (12.5 mg, 25.0 μmol) in dry DMF (1.00 mL) was successivelytreated with i-Pr₂NEt (10 μL, 6 μmol) and HBTU (11.4 mg, 30.0 μmol) at22° C. The resulting solution was stirred 0.25 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (25.0 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (3 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved in vacuo and the white solid residue redissolved in H₂Ocontaining 0.1% TFA (8.20 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from0-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 28 min was collected and lyophilized to a whitepowder (7.3 mg, 7.3 μmol; 29%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.60 (1H,brs), 8.40 (1H, brs), 8.19 (3H, brs), 4.11 (2H, brs), 3.81-3.76 (2H, m),3.55 (1H, brs), 3.49 (8H, s), 3.33 (4H, brs), 3.11 (2H, td, J=7.0, 6.0Hz), 3.02 (4H, brt, J=5.8 Hz), 1.72 (1H, brd, J=13.1 Hz), 1.67-1.49 (8H,m), 1.43 (2H, tt, J=7.6, 7.1 Hz), 1.37-1.24 (5H, m), 1.19-1.10 (3H, m).¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 165.4, 157.7 (q, J=30.7 Hz), 117.2(q, J=300 Hz), 75.3, 54.3, 53.8, 52.1, 48.7, 48.4, 40.0, 38.7, 38.4,32.8, 32.3, 32.2, 28.7, 27.4, 26.1, 25.7, 25.5, 25.4, 24.9. HRMS calcdfor C₂₉H₅₂N₆O₁₁ (M+H): 661.3767. Found: 661.3766.

Example 72-{[2-({[N-({4-[3-((2R)-2-amino-3-indol-3-ylpropanoylaminooxy)propyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of3-{4-[(Prop-2-enyloxycarbonylamino)methyl]phenyl}propanoic acid

A 500 mL Parr bottle was charged with a solution of(2E)-3-(4-cyanophenyl)prop-2-enoic acid (4.33 g, 25.0 mmol) in 2:1MeOH/28% aqueous NH₃ (300 mL) then treated with Raney Ni (5.00 g) in oneportion at 22° C. The resulting suspension was sparged with H₂ thenpressurized to 50 psi and maintained 5 h; ˜2 equiv H₂ were consumed atthis point. The vessel was then purged with N₂ and charged withadditional Raney Ni (2.5 g). The H₂ atmosphere was reestablished, andmaintained until gas uptake ceased; ˜135 psi total consumption. Thevessel was purged with N₂ and the catalyst removed by filtration throughCelite. The filter cake was exhaustively washed with 1:1 MeOH/H₂O (4×50mL) and the combined filtrates concentrated in vacuo to a white solid.

The crude amino acid (25.0 mmol theoretical) was suspended in anhydrousTHF (250 mL) then treated with i-Pr₂NEt (5.23 mL, 30.0 mmol). Allylchloroformate (3.19 mL, 30.0 mmol) was then added over 10 min and theresulting suspension stirred 1.5 h at 22° C. The now homogeneoussolution was treated with 0.1 M HCl (250 mL) then diluted with EtOAc(100 mL) with transfer to a separatory funnel. The layers separated andthe aqueous layer washed with EtOAc (2×100 mL). The combined EtOAclayers were dried over MgSO₄, filtered and concentrated in vacuo to acolorless oil that was purified by chromatography on silica (40×280 mm)using 95:5 CH₂Cl₂/MeOH (R_(f)=0.4). The main product eluted between320-420 mL, was collected and concentrated to afford a white powder(2.93 g, 11.1 mmol; 44.5%). Mp 109.5-110.5° C. ¹H NMR (CDCl₃, 600 MHz):δ 7.20 (2H, AB, J_(AB)=7.7 Hz), 7.16 (2H, AB, J_(AB)=8.0 Hz), 5.91 (1H,ddt, J=17.0, 10.7, 5.5 Hz), 5.29 (1H, brd, J=17.0 Hz), 5.20 (1H, d,J=10.2 Hz), 5.11 (1H, brs), 4.58 (2H, brd, J=4.5 Hz), 4.32 (2H, brd,J=5.7 Hz), 2.93 (2H, t, J=7.7 Hz), 2.64 (2H, t, J=7.7 Hz). ¹³C NMR(CDCl₃, 151 MHz): δ 178.2, 156.3, 139.5, 136.5, 132.8, 128.5, 127.7,117.7, 65.7, 44.8, 35.5, 30.2. HRMS calcd for C₁₄H₁₇NO₄ (M+Na):286.1050. Found: 286.1041.

Part B—Preparation ofN-{[4-(3-hydroxypropyl)phenyl]methyl}prop-2-enyloxycarboxamide

A solution of the product of Part 7A (1.32 g, 5.00 mmol) in dry THF(25.0 mL) was cooled to 0° C. and treated with LiAlH₄ (10.0 mmol; 10.0mL of a 1 M solution in THF) dropwise over 20 min using a syringe pump.The suspension was stirred 0.5 h at 0° C. then warmed to 22° C. andmaintained 2.5 h. After cooling to 0° C., excess LiAlH₄ was consumed bythe careful addition of H₂O (400 μL) and the resulting white suspensionsuccessively treated with 15% aqueous NaOH (400 μL) and H₂O (1.20 mL)then stirred for 0.5 h to a fine white slurry. The solids were removedby filtration through Celite, washed with THF (5×20 mL) and the combinedfiltrates concentrated in vacuo. The crude oil was purified bychromatography on silica (40×260 mm) using 1:1 pentane/EtOAc(R_(f)=0.2). The main product eluted between 600-800 mL, was collectedand concentrated to afford a white crystalline solid (0.795 g, 3.19mmol; 63.8%). Mp 51.5-53.5° C. ¹H NMR (CDCl₃, 600 MHz): δ 7.18 (2H, AB,J_(AB)=7.9 Hz), 7.14 (2H, AB, J_(AB)=8.2 Hz), 5.90 (1H, ddt, J=17.2,10.4, 5.7 Hz), 5.28 (1H, brd, J=17.0 Hz), 5.19 (1H, dq, J=10.5, 1.2 Hz),4.57 (2H, brd, J=4.9 Hz), 4.31 (2H, brd, J=5.8 Hz), 3.63 (2H, t, J=6.4Hz), 2.66 (2H, dd, J=7.7, 7.7 Hz), 1.87-1.82 (3H, m). ¹³C NMR (CDCl₃,151 MHz): δ 156.3, 141.1, 135.9, 132.8, 128.6, 127.5, 117.6, 65.6, 62.0,44.7, 34.1, 31.6. HRMS calcd for C₁₄H₁₉NO₃ (M+Na): 272.1257. Found:272.1263.

Part C—Preparation ofN-({4-[3-(aminooxy)propyl]phenyl}methyl)prop-2-enyloxycarboxamide,hydrochloric acid salt

A solution of the product of Part 7B (1.25 g, 5.00 mmol),2-hydroxyisoindoline-1,3-dione (0.979 g, 6.00 mmol) and PPh₃ (1.64 g,6.25 mmol) in dry THF (50.0 mL) was cooled to 0° C. and treated withDEAD (0.236 mL, 1.50 mmol) dropwise such that the orange color did notpersist. The solution was then warmed to 22° C. and treated with theremaining DEAD (0.709 mL, 4.50 mmol) dropwise over 0.75 h. The paleyellow solution thus obtained was concentrated in vacuo and directlypurified by chromatography on silica using a gradient elution from3:2→1:1 pentane/EtOAc (R_(f)=0.5 in 1:1 pentane EtOAc). The productcontaining fractions were combined and concentrated to a whitecrystalline solid that was further purified by recrystallization fromEtOAc/pentane to afford fine colorless needles (1.37 g). Despite theseefforts the material remained contaminated withethoxy-N-(ethoxycarbonylamino)carboxamide and was therefore useddirectly in the subsequent deprotection step.

The crude phthalimide (1.18 g) was dissolved in 9:1 CHCl₃/MeOH (30.0 mL)then treated with hydrazine (0.530 mL, 9.00 mmol) in one portion at 22°C. Within 5 min a white precipitate formed; after 0.25 h the reactionwas complete. The suspension was concentrated in vacuo and the resultingsolid material triturated with Et₂O (5×20 mL) then removed by filtrationthrough a scintered glass funnel. The filtrate was then treated with HCl(8.00 mmol; 2.00 mL of a 4 M solution in dioxane) and the resultingprecipitate collected. The crystalline material was further washed withH₂O and Et₂O (3×30 mL each) then dried to constant weight in vacuo(0.345 g, 1.15 mmol; 95.3%). Mp 187° C. (dec). ¹H NMR (DMSO-d₆, 600MHz): δ 11.03 (2H, brs), 7.72 (1H, brt, J=5.8 Hz), 7.16 (4H, s), 5.90(1H, dddd, J=17.0, 10.6, 5.4, 5.1 Hz), 5.27 (1H, brdd, J=17.2, 1.3 Hz),5.16 (1H, brd, J=10.2 Hz), 4.47 (2H, dt, J=5.1, 1.5 Hz), 4.14 (2H, d,J=6.1 Hz), 4.00 (2H, t, J=6.5 Hz), 2.61-2.58 (2H, m), 1.89-1.84 (2H, m).¹³C NMR (DMSO-d₆, 151 MHz): δ 156.1, 139.4, 137.4, 133.7, 128.2, 127.0,116.9, 73.4, 64.2, 43.4, 30.6, 28.9. HRMS calcd for C₁₄H₂₀N₂O₃ (M+H):265.1547. Found: 265.1550.

Part D—Preparation of(2R)—N-{3-[4-(aminomethyl)phenyl]propoxy}-2-[(tert-butoxy)-carbonylamino]-3-indol-3-ylpropanamide,trifluoroacetic acid salt

A solution of Boc-DTrp-OH (0.110 g, 0.360 mmol) in CH₂Cl₂ (3.00 mL) wassuccessively treated with HOBt (55.1 mg, 0.360 mmol), i-Pr₂NEt (125 μL,0.720 mmol) and HBTU (0.137 g, 0.360 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 7C (90.2 mg, 0.300 mmol)in one portion. The resulting solution was stirred 1 h then allvolatiles removed in vacuo. The crude hydroxamate ester was redissolvedin 2:1 MeCN/H₂O (3.00 mL) and successively treated with 17.1 mg TPPTS(30.0 μmol; 10 mol %), Et₂NH (78 μL, 0.75 mmol) and 3.4 mg Pd(OAc)₂ (15μmol; 5 mol %) at 22° C. Complete deprotection was observed within 1 h.The resulting yellow solution was diluted with H₂O containing 0.1% TFA(5.00 mL) then filtered through a 0.45 μm Acrodisk and directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 20-60% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 25 min was collected and lyophilized toa white powder (28.7 mg, 49.4 μmol; 16.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ11.10 (1H, brs), 10.81 (1H, brs), 8.13 (3H, brs), 7.57 (1H, d, J=7.7Hz), 7.36 (2H, AB, J_(AB)=8.0 Hz), 7.32 (1H, d, J=8.0 Hz), 7.26 (2H, AB,J_(AB)=8.0 Hz), 7.13 (1H, brs), 7.06 (1H, ddd, J=7.2, 7.0, 0.8 Hz), 6.98(1H, dd, J=7.5, 7.2 Hz), 6.93 (1H, brd, J=7.7 Hz), 4.02 (1H, td, J=8.0,6.5 Hz), 4.01-3.98 (2H, m), 3.70-3.59 (2H, m), 3.00 (1H, ABX,J_(AB)=14.1 Hz, J_(AX)=6.1 Hz), 2.90 (1H, ABX, J_(AB)=14.5 Hz,J_(BX)=8.5 Hz), 2.66-2.60 (2H, m), 1.73 (2H, brs), 1.33 (9H, s). ¹³C NMR(DMSO-d₆, 151 MHz): δ 168.6, 155.0, 142.1, 136.0, 131.3, 128.8, 128.6,127.2, 123.7, 120.8, 118.4, 118.1, 111.2, 109.8, 78.0, 74.0, 52.9, 42.1,31.0, 29.3, 28.1, 27.6. HRMS calcd for C₂₆H₃₄N₄O₄ (M+H): 467.2653.Found: 467.2649.

Part E—Preparation of2-{[2-({[N-({4-[3-((2R)-2-amino-3-indol-3-ylpropanoylaminooxy)-propyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)-ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (18.5 mg, 30.0 μmol), HOBt (4.6 mg, 30.0 μmol) and the product ofPart 7D (14.5 mg, 25.0 μmol) in dry DMF (1.00 mL) was successivelytreated with i-Pr₂NEt (10 μL, 6 μmol) and HBTU (11.4 mg, 30.0 μmol) at22° C. The resulting solution was stirred 0.25 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (25.0 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (3 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved in vacuo and the white solid residue redissolved in H₂Ocontaining 0.1% TFA (8.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from10-50% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 19 min was collected and lyophilized to a whitepowder (3.4 mg, 3.1 μmol; 12.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.50(1H, brs), 11.01 (1H, brs), 8.87 (1H, brs), 8.26 (3H, brs), 7.59 (1H, d,J=7.8 Hz), 7.36 (1H, d, J=8.1 Hz), 7.19 (2H, AB, J_(AB)=8.1 Hz), 7.14(2H, AB, J_(AB)=8.0 Hz), 7.09 (1H, dd, J=7.6, 7.4 Hz), 7.01 (1H, dd,J=7.5, 7.3 Hz), 6.50 (1H, brs), 4.31 (2H, brd, J=5.4 Hz), 4.19 (2H,brs), 3.73 (1H, brs), 3.64 (1H, ABXY, J_(AB)=9.6 Hz, J_(AX)=6.6 Hz,J_(AY)=6.4 Hz), 3.57 (1H, ABXY, J_(AB)=9.6 Hz, J_(BX)=6.4 Hz, J_(BY)=6.3Hz), 3.49 (8H, s), 3.35 (4H, brs), 3.17 (1H, ABX, J_(AB)=14.3 Hz,J_(AX)=7.2 Hz), 3.09 (1H, ABX, J_(AB)=14.3 Hz, J_(BX)=6.8 Hz), 3.03 (4H,brt, J=5.2 Hz), 2.55 (2H, dd, J=7.7, 7.6 Hz), 1.70-1.63 (2H, m). ¹³C NMR(DMSO-d₆, 151 MHz): δ 172.7, 165.0, 157.7 (q, J=30.7 Hz), 140.2, 136.2,135.7, 128.3, 127.4, 126.8, 124.6, 121.2, 118.5, 118.2, 117.2 (q, J=301Hz), 111.5, 106.7, 74.6, 54.3, 53.9, 52.2, 51.0, 48.7, 42.1, 30.8, 29.1,27.2. HRMS calcd for C₃₅H₄₇N₇O₁₁ (M+H): 742.3406. Found: 742.3401.

Example 82-{[2-({[N-({4-[3-((2R)-2-amino-4-phenylbutanoylaminooxy)propyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-{3-[4-(aminomethyl)phenyl]propoxy}-2-[(tert-butoxy)carbonylamino]-4-phenylbutanamide,trifluoroacetic acid salt

A solution of Boc-DHfe-OH (0.101 g, 0.360 mmol) in CH₂Cl₂ (3.00 mL) wassuccessively treated with HOBt (55.1 mg, 0.360 mmol), i-Pr₂NEt (125 μL,0.720 mmol) and HBTU (0.137 g, 0.360 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 7C (90.2 mg, 0.300 mmol)in one portion. The resulting solution was stirred 1 h then allvolatiles removed in vacuo. The crude hydroxamate ester was redissolvedin 2:1 MeCN/H₂O (3.00 mL) and successively treated with 17.1 mg TPPTS(30.0 μmol; 10 mol %), Et₂NH (78 μL, 0.75 mmol) and 3.4 mg Pd(OAc)₂ (15μmol; 5 mol %) at 22° C. Complete deprotection was observed within 1 h.The resulting yellow solution was diluted with H₂O containing 0.1% TFA(5.00 mL) then filtered through a 0.45 μm Acrodisk and directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 30-70% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 25 min was collected and lyophilized toa white powder (48.2 mg, 86.8 μmol; 28.9%). ¹H NMR (DMSO-d₆, 600 MHz): δ11.11 (1H, brs), 8.12 (3H, brs), 7.34 (2H, AB, J_(AB)=8.1 Hz), 7.28-7.25(4H, m), 7.18-7.16 (3H, m), 7.06 (1H, brd, J=7.8 Hz), 4.00-3.96 (2H, m),3.77-3.70 (3H, m), 2.67 (2H, t, J=7.6 Hz), 2.62-2.57 (1H, m), 2.52-2.47(1H, m), 1.83-1.78 (4H, m), 1.38 (9H, s). ¹³C NMR (DMSO-d₆, 75 MHz): δ168.8, 155.3, 142.1, 141.2, 131.4, 128.8, 128.6, 128.3, 125.8, 76.1,74.2, 51.9, 42.0, 33.5, 31.5, 31.0, 29.4, 28.1. HRMS calcd forC₂₅H₃₅N₃O₄ (M+H): 442.2700. Found: 442.2698.

Part B—Preparation of2-{[2-({[N-({4-[3-((2R)-2-amino-4-phenylbutanoylaminooxy)-propyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)-ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (18.5 mg, 30.0 μmol), HOBt (4.6 mg, 30.0 μmol) and the product ofPart 8A (13.9 mg, 25.0 μmol) in dry DMF (1.00 mL) was successivelytreated with i-Pr₂NEt (10 μL, 6 μmol) and HBTU (11.4 mg, 30.0 μmol) at22° C. The resulting solution was stirred 0.25 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (25.0 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (3 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved in vacuo and the white solid residue redissolved in H₂Ocontaining 0.1% TFA (8.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from10-50% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 21 min was collected and lyophilized to a whitepowder (10.5 mg, 9.92 μmol; 39.7%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.74(1H, brs), 8.89 (1H, brt, J=5.8 Hz), 8.35 (3H, brs), 7.30 (2H, dd,J=7.6, 7.3 Hz), 7.22-7.16 (8H, m), 4.30 (2H, brd, J=5.5 Hz), 4.22 (2H,s), 3.83 (2H, dd, J=6.4, 6.1 Hz), 3.67 (1H, brs), 3.49 (8H, s), 3.37(4H, brt, J=5.5 Hz), 3.04 (4H, brt, J=5.7 Hz), 2.66 (2H, dd, J=7.9, 7.6Hz), 2.58 (2H, dd, J=8.4, 8.2 Hz), 2.01-1.92 (2H, m), 1.87-1.82 (2H, m).¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 165.1, 164.6, 157.9 (q, J=31.7 Hz),140.3, 140.2, 135.8, 128.5, 128.3, 128.0, 127.4, 126.2, 117.0 (q, J=299Hz), 74.8, 54.3, 53.9, 52.2, 50.3, 48.6, 42.1, 32.8, 30.9, 30.4, 29.4.HRMS calcd for C₃₄H₄₈N₆O₁₁ (M+H): 717.3454. Found: 717.3446.

Example 92-({2-[({N-[6-((2R)-2-amino-4-phenylbutanoylaminooxy)hexyl]carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)amino)aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-(6-aminohexyloxy)-2-[(tert-butoxy)carbonylamino]-4-phenylbutanamide,trifluoroacetic acid salt

A solution of Boc-DHfe-OH (0.101 g, 0.360 mmol) in CH₂Cl₂ (3.00 mL) wassuccessively treated with HOBt (55.1 mg, 0.360 mmol), i-Pr₂NEt (125 μL,0.720 mmol) and HBTU (0.137 g, 0.360 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 4C (75.8 mg, 0.300 mmol)in one portion. The resulting solution was stirred 1 h then allvolatiles removed in vacuo. The crude hydroxamate ester was redissolvedin 2:1 MeCN/H₂O (3.00 mL) and successively treated with 17.1 mg TPPTS(30.0 μmol; 10 mol %), Et₂NH (78 μL, 0.75 mmol) and 3.4 mg Pd(OAc)₂ (15μmol; 5 mol %) at 22° C. Complete deprotection was observed within 1 h.The resulting yellow solution was diluted with H₂O containing 0.1% TFA(5.00 mL) then filtered through a 0.45 μm Acrodisk and directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 10-50% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 32 min was collected and lyophilized toa white powder (37.8 mg, 74.5 μmol; 24.8%). A small amount of TPPTS canbe detected in the ¹H NMR spectrum. ¹H NMR (DMSO-d₆, 600 MHz): δ 11.07(1H, brs), 7.77 (3H, brs), 7.26 (2H, dd, J=7.6, 7.6 Hz), 7.18-7.15 (3H,m), 7.04 (1H, brd, J=7.6 Hz), 3.77-3.70 (3H, m), 2.78-2.73 (2H, m),2.62-2.57 (1H, m), 2.52-2.47 (1H, m), 1.82-1.75 (2H, m), 1.54-1.49 (4H,m), 1.38 (9H, s), 1.38-1.27 (4H, m). ¹³C NMR (DMSO-d₆, 151 MHz): δ168.7, 158.2 (q, J=32.0 Hz), 155.2, 141.23, 128.3, 128.2, 125.8, 116.9(q, J=293 Hz), 78.0, 74.8, 51.9, 38.7, 33.6, 31.5, 28.1, 27.2, 26.8,25.5, 24.8. HRMS calcd for C₂₁H₃₅N₃O₄ (M+H): 394.2700. Found: 394.2698.

Part B—Preparation of2-({2-[({N-[6-((2R)-2-amino-4-phenylbutanoylaminooxy)hexyl]-carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)-amino)aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (18.5 mg, 30.0 μmol), HOBt (4.6 mg, 30.0 μmol) and the product ofPart 9A (12.7 mg, 25.0 μmol) in dry DMF (1.00 mL) was successivelytreated with i-Pr₂NEt (10 μL, 6 μmol) and HBTU (11.4 mg, 30.0 μmol) at22° C. The resulting solution was stirred 0.25 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (25.0 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (3 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved in vacuo and the white solid residue redissolved in H₂Ocontaining 0.1% TFA (8.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from0-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 27 min was collected and lyophilized to a whitepowder (10.4 mg, 10.3 μmol; 41.2%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.64(1H, brs), 8.41 (1H, brt, J=5.3 Hz), 8.31 (2H, brs), 7.30 (2H, dd,J=7.6, 7.5 Hz), 7.21 (1H, dd, J=7.4, 7.4 Hz), 7.18 (2H, d, J=7.2 Hz),4.13 (2H, brs), 3.84-3.76 (2H, m), 3.64 (1H, brs), 3.49 (8H, s), 3.34(4H, brt, J=4.9 Hz), 3.10 (2H, td, J=6.8, 6.0 Hz), 3.03 (4H, brt, J=5.7Hz), 2.58 (2H, dd, J=8.4, 8.0 Hz), 2.01-1.93 (2H, m), 1.59-1.54 (2H, m),1.45-1.40 (2H, m), 1.38-1.25 (4H, m). ¹³C NMR (DMSO-d₆, 151 MHz): δ172.7, 165.0, 164.3, 157.7 (q, J=31.4 Hz), 140.1, 128.5, 128.0, 126.2,117.0 (q, J=300 Hz), 75.4, 54.3, 53.9, 52.1, 50.2, 48.6, 38.7, 32.8,30.3, 28.7, 27.4, 26.1, 24.9. HRMS calcd for C₃₀H₄₈N₆O₁₁ (M+H):669.3454. Found: 669.3446.

Example 102-[(2-{[(N-{[4-((2R)-2-amino-4-methylpentanoylaminooxy)phenyl]methyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

Part A—Preparation ofN-{[4-(aminooxy)phenyl]methyl}prop-2-enyloxycarboxamide, hydrochloricacid salt

A solution of N-[(4-hydroxyphenyl)methyl]prop-2-enyloxycarboxamide (2.07g, 10.0 mmol; Imamura, H.; Ohtake, N.; Shimizu, A.; Jona, H.; Sato, H.;Nagano, R.; Ushijima, R.; Yamada, K.; Hashizume, T.; Morishima, H.Bioorg. Med. Chem. Lett. 2000, 10(2), 109-113.) in dry MeOH (20.1 mL)was cooled to 0° C. and treated with KOt-Bu (1.12 g, 10.0 mmol) in oneportion. The resulting pale pink solution was stirred 0.25 h, thenwarmed to 22° C., maintained 0.25 h and concentrated in vacuo. Thesolids were redissolved in DMF (13.0 mL), cooled to 0° C. then treatedwith freshly prepared amino 2,4,6-trimethylbenzenesulfonate (10.0 mmol;6.00 mL of a 1.67 M solution in DMF; (a) Carpino, L. A. J. Am. Chem.Soc. 1960, 82, 3133. (b) Krause, J. G. Synthesis 1972, 3, 140. (c)Suits, J. Z.; Applequist, D. E.; Swart, D. J. J. Org. Chem. 1983, 48,5120.) dropwise over 5 min; additional DMF (2×0.50 mL) was used toquantitate the transfer. After 0.5 h, the resulting solution was dilutedwith H₂O (100 mL) with transfer to a separatory funnel, then washed withEt₂O (5×50 mL). The combined Et₂O washes were dried over MgSO₄, filteredthen treated with HCl (4.00 mmol; 1.00 mL of a 4 M solution in dioxane)at 22° C. The resulting plate-like crystals were collected on ascintered glass funnel of fine porosity, washed with Et₂O and pentane(5×20 mL each) then dried to constant weight on the funnel (0.597 g,2.31 mmol; 23.1%). ¹H NMR (DMSO-d₆, 300 MHz): δ 7.74 (1H, brt, J=6.0Hz), 7.25 (2H, AA′BB′, J_(AB)=8.8 Hz, J_(AA′)=2.5 Hz), 7.14 (2H, AA′BB′,J_(AB)=8.8 Hz, J_(BB′)=2.5 Hz), 5.90 (1H, ddt, J=17.2, 10.5, 5.4 Hz),5.26 (1H, dq, J=17.3, 1.5 Hz), 5.16 (1H, dq, J=10.4, 1.4 Hz), 4.47 (2H,dt, J=5.3, 1.5 Hz), 4.13 (2H, brd, J=6.1 Hz). ¹³C NMR (DMSO-d₆, 151MHz): δ 156.1, 156.0, 135.3, 133.7, 128.2, 116.9, 114.3, 64.3, 43.1.HRMS calcd for C₁₁H₁₄N₂O₃ (M+H): 223.1077. Found: 223.1079.

Part B—Preparation of(2R)—N-[4-(aminomethyl)phenoxy]-2-[(tert-butoxy)carbonylamino]-4-methylpentanamide,trifluoroacetic acid salt

A solution of Boc-DLeu-OH (0.139 g, 0.600 mmol) and HOBt (91.9 mg, 0.600mmol) in DMF (5.00 mL) was successively treated with i-Pr₂NEt (209 μL,1.20 mmol) and HBTU (0.228 g, 0.600 mmol) at 22° C. After 10 min, thesolution was treated with the product of Part 10A (0.129 g, 0.500 mmol)in one portion. The resulting solution was stirred 17 h then treatedwith additional HBTU (56.9 mg, 0.150 mmol) to complete conversion. After1 h, the solution was partitioned between EtOAc and 0.1 M citric acid(30 mL each) then transferred to a separatory funnel. The layersseparated and the aqueous solution washed with EtOAc (2×30 mL). Thecombined EtOAc layers were successively washed with 0.1 M citric acidand saturated aqueous solutions of NaHCO₃ and NaCl (3×30 mL each) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The crude hydroxamate ester (0.500 mmol theoretical) was dissolved in2:1 MeCN/H₂O (5.00 mL) and successively treated with 28.4 mg TPPTS (50.0μmol; 10 mol %), Et₂NH (129 μL, 1.25 mmol) and 5.6 mg Pd(OAc)₂ (25.0μmol; 5 mol %) at 22° C. Complete deprotection was observed within 0.5h. The resulting amber solution was diluted with H₂O containing 0.1% TFA(3.00 mL) then filtered through a 0.45 μm Acrodisk and directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 10-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 23 min was collected and lyophilized toa white powder (71.3 mg, 0.153 mmol; 30.6%). ¹H NMR (DMSO-d₆, 300 MHz):δ 12.11 (1H, brs), 8.10 (3H, brs), 7.36 (2H, AB, J_(AB)=8.5 Hz), 7.17(1H, brd, J=7.8 Hz), 7.05 (2H, AB, J_(AB)=8.6 Hz), 4.00-3.90 (3H, m),1.66-1.36 (3H, m), 1.41 (9H, s), 0.90 (3H, d, J=6.4 Hz), 0.86 (3H, d,J=6.5 Hz). ¹³C NMR (DMSO-d₆, 75 MHz): δ 169.8, 159.6, 155.5, 130.2,127.6, 112.8, 78.2, 50.7, 41.7, 28.1, 24.2, 22.6, 21.7. HRMS calcd forC₁₈H₂₉N₃O₄(M+H−NH₃): 335.1965. Found: 335.1969.

Part C—Preparation of2-[(2-{[(N-{[4-((2R)-2-amino-4-methylpentanoylaminooxy)-phenyl]methyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}-ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (67.9 mg, 0.110 mmol), HOBt (16.8 mg, 0.110 mmol) and the productof Part 10B (46.5 mg, 0.100 mmol) in dry DMF (2.00 mL) was successivelytreated with i-Pr₂NEt (38 μL, 0.22 mmol) and HBTU (41.7 mg, 0.110 mmol)at 22° C. The resulting solution was stirred 0.5 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (0.110 mmol theoretical) was dissolved indioxane (1.00 mL) then successively treated with H₂O (10 μL) and HCl(4.00 mmol; 1.00 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 15 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed in vacuo and the white solid residue redissolvedin H₂O containing 0.1% TFA (6.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from0-22% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 18 min was collected and lyophilized to a whitepowder (30.8 mg, 31.8 μmol; 31.8%). ¹H NMR (DMSO-d₆, 600 MHz): δ 12.70(1H, brs), 8.90 (1H, brs), 8.41 (3H, brs), 7.26 (2H, AB, J_(AB)=8.4 Hz),7.03 (2H, AB, J_(AB)=8.1 Hz), 4.30 (2H, brd, J=5.2 Hz), 4.20 (2H, s),3.81 (1H, brs), 3.50 (8H, s), 3.36 (4H, brt, J=5.4 Hz), 3.04 (4H, brt,J=5.8 Hz), 1.64 (3H, brs), 0.95 (3H, brd, J=5.5 Hz), 0.92 (3H, brd,J=5.5 Hz). ¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 166.4, 164.7, 158.2,158.0 (q, J=30.7 Hz), 132.8, 128.7, 117.1 (q, J=300 Hz), 113.0, 54.3,53.9, 52.2, 49.0, 48.7, 41.7, 40.0, 23.8, 22.2, 22.0. HRMS calcd forC₂₇H₄₂N₆O₁₁ (M+H): 627.2986. Found: 627.2989.

Example 112-[(2-{[(N-{[4-((2R)-2-amino-4-phenylbutanoylaminooxy)phenyl]methyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-[4-(aminomethyl)phenoxy]-2-[(tert-butoxy)carbonylamino]-4-phenylbutanamide,trifluoroacetic acid salt

A solution of Boc-DHfe-OH (0.168 g, 0.600 mmol) and HOBt (91.9 mg, 0.600mmol) in DMF (5.00 mL) was successively treated with i-Pr₂NEt (209 μL,1.20 mmol) and HBTU (0.228 g, 0.600 mmol) at 22° C. After 10 min, thesolution was treated with the product of Part 10A (0.129 g, 0.500 mmol)in one portion. The resulting solution was stirred 17 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous solution washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The crude hydroxamate ester (0.500 mmol theoretical) was dissolved in2:1 MeCN/H₂O (5.00 mL) and successively treated with 28.4 mg TPPTS (50.0μmol; 10 mol %), Et₂NH (129 μL, 1.25 mmol) and 5.6 mg Pd(OAc)₂ (25.0μmol; 5 mol %) at 22° C. Complete deprotection was observed within 0.5h. The resulting amber solution was diluted with H₂O containing 0.1% TFA(3.00 mL) then lyophilized. The solid was redissolved in 10:1 H₂O/MeCN(8.00 ml), filtered through a 0.45 μm Acrodisk and directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 20-50% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 17 min was collected and lyophilized toa white powder (0.157 g, 0.305 mmol; 61.0%). ¹H NMR (DMSO-d₆, 600 MHz):δ 12.11 (1H, brs), 8.10 (3H, brs), 7.36 (2H, AB, J_(AB)=8.5 Hz), 7.34(1H, brd, J=7.5 Hz), 7.28 (2H, dd, J=7.7, 7.5 Hz), 7.20 (2H, AB,J_(AB)=7.5 Hz), 7.18 (1H, t, J=7.2 Hz), 7.06 (2H, AB, J_(AB)=8.5 Hz),3.96 (2H, brd, J=5.1 Hz), 3.90-3.87 (1H, m), 2.70-2.65 (1H, m),2.59-2.54 (1H, m), 1.93-1.87 (2H, m), 1.43 (9H, s). ¹³C NMR (DMSO-d₆,151 MHz): δ 169.6, 159.6, 155.5, 141.0, 130.2, 128.3, 127.7, 125.9,112.8, 78.3, 52.2, 41.6, 32.8, 31.5, 28.2. HRMS calcd for C₂₂H₂₉N₃O₄(M+H): 400.2231. Found: 400.2241.

Part B—Preparation of2-[(2-{[(N-{[4-((2R)-2-amino-4-phenylbutanoylaminooxy)-phenyl]methyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}-ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (47.9 mg, 77.6 μmol), HOBt (10.9 mg, 71.1 μmol) and the product ofPart 11A (33.2 mg, 64.7 μmol) in dry DMF (1.29 mL) was successivelytreated with i-Pr₂NEt (25 μL, 0.14 mmol) and EDC (13.6 mg, 71.1 μmol) at22° C. The resulting solution was stirred 20 h then partitioned betweenEtOAc and 0.1 M citric acid (30 mL each) with transfer to a separatoryfunnel. The layers separated and the aqueous layer washed with EtOAc(2×30 mL). The combined EtOAc layers were successively washed with 0.1 Mcitric acid, 0.1 M NaOH and saturated aqueous NaCl (3×30 mL each) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The protected conjugate (64.7 μmol theoretical) was dissolved in dioxane(0.650 mL) then successively treated with H₂O (6 μL) and HCl (2.60 mmol;0.650 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18.5 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved under a stream of N₂ and the white solid residue redissolved inH₂O containing 0.1% TFA (8.00 mL) then directly purified by HPLC on aPhenomenex Luna C18 column (21.2×250 mm) using a 1%/min gradient from0-30% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min. The mainproduct peak eluting at 23 min was collected and lyophilized to a whitepowder (29.4 mg, 28.9 μmol; 44.7%). ¹H NMR (DMSO-d₆, 600 MHz): δ 12.79(1H, brs), 8.92 (1H, brs), 8.56 (3H, brs), 7.32 (2H, dd, J=7.8, 7.1 Hz),8.27 (2H, AB, J_(AB)=8.4 Hz), 7.23-7.21 (3H, m), 7.06 (2H, d, J=7.6 Hz),4.31 (2H, brd, J=5.0 Hz), 4.23 (2H, s), 3.50 (8H, s), 3.38 (4H, brs),3.05 (4H, brt, J=5.4 Hz), 2.67 (2H, brs), 2.09 (2H, brs). ¹³C NMR(DMSO-d₆, 151 MHz): δ 172.7, 166.1, 164.6, 158.3, 158.2 (q, J=32.9 Hz),140.2, 132.8, 128.7, 128.6, 128.1, 126.3, 116.9 (q, J=299 Hz), 113.0,54.3, 53.9, 52.2, 50.4, 48.7, 41.8, 32.9, 30.5. HRMS calcd forC₃₁H₄₂N₆O₁₁ (M+H): 675.2984. Found: 675.2997.

Example 122-[(2-{[(N-{[4-((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)phenyl]methyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-[4-(aminomethyl)phenoxy]-2-[(tert-butoxy)carbonylamino]-3-(2-naphthyl)propanamide,trifluoroacetic acid salt

A solution of Boc-DNal-OH (0.189 g, 0.600 mmol) and HOBt (91.9 mg, 0.600mmol) in DMF (5.00 mL) was successively treated with i-Pr₂NEt (209 μL,1.20 mmol) and HBTU (0.228 g, 0.600 mmol) at 22° C. After 10 min, thesolution was treated with the product of Part 10A (0.129 g, 0.500 mmol)in one portion. The resulting solution was stirred 17 h then treatedwith additional HBTU (56.9 mg, 0.150 mmol) to complete conversion. After1 h, the solution was partitioned between EtOAc and 0.1 M citric acid(30 mL each) then transferred to a separatory funnel. The layersseparated and the aqueous solution washed with EtOAc (2×30 mL). Thecombined EtOAc layers were successively washed with 0.1 M citric acidand saturated aqueous solutions of NaHCO₃ and NaCl (3×30 mL each) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The crude hydroxamate ester (0.500 mmol theoretical) was dissolved in2:1 MeCN/H₂O (5.00 mL) and successively treated with 28.4 mg TPPTS (50.0μmol; 10 mol %), Et₂NH (129 μL, 1.25 mmol) and 5.6 mg Pd(OAc)₂ (25.0μmol; 5 mol %) at 22° C. Complete deprotection was observed within 0.5h. The resulting amber solution was diluted with H₂O containing 0.1% TFA(3.00 mL) then lyophilized. The solid was redissolved in 1:1 H₂O/MeCN(8.00 ml), filtered through a 0.45 μm Acrodisk and directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 30-60% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 12 min was collected and lyophilized toa white powder (0.115 g, 0.209 mmol; 41.9%). ¹H NMR (DMSO-d₆, 600 MHz):δ 12.08 (1H, s), 8.10 (3H, brs), 7.89 (1H, d, J=7.5 Hz), 7.86 (1H, d,J=8.5 Hz), 7.83 (1H, d, J=7.5 Hz), 7.76 (1H, s), 7.51-7.47 (2H, m), 7.45(1H, d, J=8.0 Hz), 7.40 (1H, brd, J=7.7 Hz), 7.21 (2H, AB, J_(AB)=8.3Hz), 6.86 (2H, AB, J_(AB)=8.5 Hz), 4.28-4.24 (1H, m), 3.92 (2H, brs),3.14 (1H, ABX, J_(AB)=13.6 Hz, J_(AX)=6.7 Hz), 3.06 (1H, ABX,J_(AB)=13.3 Hz, J_(BX)=8.8 Hz), 1.35 (9H, s). ¹³C NMR (DMSO-d₆, 151MHz): δ 168.8, 159.4, 155.3, 135.1, 132.9, 131.9, 130.1, 127.6, 127.6,127.6, 127.5, 127.4, 126.0, 125.5, 112.7, 78.3, 53.8, 41.6, 36.9, 28.1.HRMS calcd for C₂₅H₂₉N₃O₄ (M+H−NH₃): 419.1965. Found: 419.1967.

Part B—Preparation of2-[(2-{[(N-{[4-((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)-phenyl]methyl}carbamoyl)methyl]{2-[bis(carboxymethyl)amino]ethyl}amino}-ethyl)(carboxymethyl)amino]aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (67.9 mg, 0.110 mmol), HOBt (16.8 mg, 0.110 mmol) and the productof Part 12A (54.9 mg, 0.100 mmol) in dry DMF (2.00 mL) was successivelytreated with i-Pr₂NEt (38 μL, 0.22 mmol) and HBTU (41.7 mg, 0.110 mmol)at 22° C. The resulting solution was stirred 0.5 h then partitionedbetween EtOAc and 0.1 M citric acid (30 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×30 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid and saturated aqueous solutions of NaHCO₃ andNaCl (3×30 mL each) then dried over MgSO₄, filtered and concentrated invacuo to a colorless oil which was used without further purification inthe subsequent deprotection step.

The protected conjugate (0.100 mmol theoretical) was dissolved indioxane (1.00 mL) then successively treated with H₂O (10 μL) and HCl(4.00 mmol; 1.00 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 15 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O containing 0.1% TFA (8.00 mL) then directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 5-30% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 20 min was collected and lyophilized toa white powder (40.5 mg, 38.5 μmol; 38.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ12.49 (1H, brs), 8.84 (1H, brt, J=5.1 Hz), 8.63 (2H, brs), 7.98-7.95(2H, m), 7.89-7.86 (1H, m), 7.77 (1H, brs), 7.57-7.54 (2H, m), 7.45 (1H,brs), 6.85 (2H, AB, J_(AB)=8.2 Hz), 6.50 (2H, AB, J_(AB)=7.6 Hz), 4.20(2H, s), 4.21-4.15 (3H, m), 3.51 (8H, s), 3.38 (4H, brt, J=5.5 Hz),3.35-3.31 (1H, m), 3.26-3.22 (1H, m), 3.05 (4H, brt, J=5.7 Hz). ¹³C NMR(DMSO-d₆, 151 MHz): δ 172.7, 156.2, 164.6, 158.1 (q, J=31.8 Hz), 157.8,133.0, 132.4, 132.3, 132.2, 128.4, 128.3, 128.3, 127.6, 127.6, 127.3,126.3, 126.0, 117.1 (q, J=299 Hz), 112.6, 54.3, 53.8, 52.2, 51.6, 48.7,41.7, 37.0. HRMS calcd for C₃₄H₄₂N₆O₁₁ (M+H): 711.2986. Found: 711.2985.

Example 132-{[2-({[N-({4-[2-((2R)-2-amino-4-methylpentanoylaminooxy)ethyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation ofN-methoxy-N-methyl(4-{[(phenylmethoxy)carbonylamino]-methyl}phenyl)carboxamide

A solution of 4-{[(phenylmethoxy)carbonylamino]methyl}benzoic acid (3.99g, 14.0 mmol; Groves, K.; Wilson, A. J.; Hamilton, A. D. J. Am. Chem.Soc. 2004, 126(40), 12833-12842.) and HOBt (2.57 g, 16.8 mmol) in dryDMF (70.0 mL) was successively treated with i-Pr₂NEt (4.87 mL, 28.0mmol) and EDC (3.22 g, 16.8 mmol) at 22° C. After 0.25 h, the solutionwas treated with methoxymethylamine hydrochloride (1.64 g, 16.8 mmol) inone portion. The resulting mixture was stirred 1 h then partitionedbetween EtOAc and 0.1 M citric acid (100 mL each) with transfer to aseparatory funnel. The layers separated and the aqueous layer washedwith EtOAc (2×50 mL). The combined EtOAc layers were successively washedwith 0.1 M citric acid, 0.1 M NaOH and saturated aqueous NaCl (3×50 mLeach) then dried over MgSO₄, filtered and concentrated in vacuo.Purification by chromatography on silica (40×250 mm) using a gradientelution from 1:1→3:7 pentane/EtOAc (R_(f)=0.3 in 1:1 hexanes/EtOAc)afforded pure material as a colorless oil (3.94 g, 12.0 mmol; 85.9%). ¹HNMR (CDCl₃, 300 MHz): δ 7.63 (2H, AA′BB′, J_(AB)=8.3 Hz, J_(AA′)=1.9Hz), 7.36-7.27 (7H, m), 5.20 (1H, brs), 5.13 (2H, s), 4.40 (2H, brd,J=6.0 Hz), 3.52 (3H, s), 3.33 (3H, s). ¹³C NMR (CDCl₃, 75 MHz): δ 169.5,156.4, 141.1, 136.4, 133.2, 128.6, 128.5, 128.1, 128.1, 126.9, 66.9,61.0, 44.8, 33.7. HRMS calcd for C₁₈H₂₀N₂O₄: 329.1496. Found: 329.1497.

Part B—Preparation ofN-[(4-acetylphenyl)methyl](phenylmethoxy)carboxamide

A solution of the product of Part 13A (3.28 g, 10.0 mmol) in dry THF(100 mL) was cooled to 0° C. and treated with MeLi (30.0 mmol; 10.2 mLof a 2.94 M solution in Et₂O) dropwise over 0.25 h; during the additiona heavy white precipitate formed. After 0.5 h, the resulting suspensionwas treated with a solution of conc. HCl in absolute EtOH (5:95 v/v; 100mL) then diluted with Et₂O and saturated aqueous NaCl (100 mL each) withtransfer to a separatory funnel. The layers separated and the aqueouslayer washed with Et₂O (2×50 mL). The combined Et₂O layers were furtherwashed with saturated aqueous NaCl (3×100 mL) then dried over MgSO₄,filtered and concentrated in vacuo. The crude material was purified bychromatography on silica (40×300 mm) using 1:1 hexanes/EtOAc. The mainproduct eluted between 300-500 mL, was collected and concentrated to anamorphous white powder that was recrystallized from Et₂O/pentane toafford fine colorless needles (1.57 g, 5.54 mmol; 55.6%). Mp101.0-103.0° C. ¹H NMR (CDCl₃, 300 MHz): δ 7.90 (2H, AB, J_(AB)=8.3 Hz),7.34 (7H, brs), 5.22 (1H, brs), 5.13 (2H, s), 4.40 (2H, brd, J=6.2 Hz),2.57 (3H, s). ¹³C NMR (DMSO-d₆, 151 MHz): δ 197.6, 156.4, 143.9, 136.4,136.3, 128.7, 128.5, 128.2, 128.1, 127.4, 67.0, 44.7, 26.6. HRMS calcdfor C₁₇H₁₇NO₃ (M+H): 284.1281. Found: 284.1280.

Part C—Preparation of Methyl2-(4-{[(phenylmethoxy)carbonylamino]methyl}phenyl)acetate

A solution of the product of Part 13B (1.24 g, 4.38 mmol) in 3:1MeOH/HC(OMe)₃ (28.0 mL) was successively treated with AgNO₃ (1.56 g,9.18 mmol) and I₂ (1.17 g, 4.61 mmol) at 22° C. The resulting solutionwas warmed to 68° C. and maintained at reflux for 2 h. After cooling to22° C., the suspension was filtered through a scintered glass funnel andthe filtrate partitioned between Et₂O and H₂O (50 mL each) with transferto a separatory funnel. The layers separated and the aqueous layerwashed with Et₂O (2×50 mL). The combined Et₂O layers were dried overMgSO₄, filtered and concentrated in vacuo to a white solid that was usedwithout further purification in the subsequent reduction step. ¹H NMR(CDCl₃, 300 MHz): δ 7.87-7.80 (5H, m), 7.23 (4H, s), 5.13 (2H, s), 5.04(1H, brs), 4.36 (2H, brd, J=5.9 Hz), 3.68 (3H, s), 3.60 (2H, s).

Part D—Preparation ofN-{[4-(2-hydroxyethyl)phenyl]methyl}(phenylmethoxy)carboxamide

A solution of the product of Part 13C (1.20 g, 3.83 mmol) in dry THF(38.3 mL) was cooled to 0° C. and treated with LiAlH₄ (3.83 mmol; 3.83mL of a 1 M solution in THF) dropwise over 10 min. The resultingsolution was stirred 0.25 h at 0° C. to ensure complete reduction.Excess LiAlH₄ was consumed by the careful addition of H₂O (145 μL). Theresulting white suspension was successively treated with 15% aqueousNaOH (145 μL) and H₂O (435 μL) then stirred for 0.25 h to a fine whiteslurry. The resulting mixture was filtered through a pad of Celite andconcentrated in vacuo. The crude oil was purified by chromatography onsilica using 1:1 hexanes/EtOAc to afford a white solid (0.670 g, 2.35mmol; 61.3%). ¹H NMR (CDCl₃, 600 MHz): δ 7.36-7.29 (5H, m), 7.23 (2H,AB, J_(AB)=7.3 Hz), 7.19 (2H, AB, J_(AB)=7.7 Hz), 5.13 (2H, s), 5.03(1H, brs), 4.36 (2H, brd, J=5.5 Hz), 3.84 (2H, t, J=6.6 Hz), 2.85 (2H,t, J=6.6 Hz), 1.46 (1H, brs). HRMS calcd for C₁₇H₁₉NO₃ (M+Na): 308.1257.Found: 308.1257.

Part E—Preparation ofN-({4-[2-(1,3-dioxoisoindolin-2-yloxy)ethyl]phenyl}methyl)-(phenylmethoxy)carboxamide

A solution of the product of Part 13D (0.300 g, 1.05 mmol),2-hydroxyisoindoline-1,3-dione (0.206 g, 1.26 mmol) and PPh₃ (0.414 g,1.58 mmol) in dry THF (10.5 mL) was cooled to 0° C. and treated withDEAD (0.224 mL, 1.42 mmol) dropwise such that the orange color did notpersist. The pale yellow solution thus obtained was immediately warmedto 22° C., concentrated in vacuo and directly purified by chromatographyon silica using a gradient elution from 2:1→1:1 hexanes/EtOAc (R_(f)=0.5in 1:1 hexanes/EtOAc). The product containing fractions were combinedand concentrated to a white crystalline solid (0.354 g, 0.822 mmol;78.2%). ¹H NMR (CDCl₃, 600 MHz): δ 7.83-7.80 (2H, m), 7.74-7.72 (2H, m),7.36-7.29 (5H, m), 7.26 (2H, AB, J_(AB)=8.0 Hz), 7.21 (2H, AB,J_(AB)=7.5 Hz), 5.13 (2H, s), 4.98 (1H, brs), 4.42 (2H, t, J=7.3 Hz),4.33 (2H, brd, J=5.5 Hz), 3.12 (2H, t, J=7.3 Hz). HRMS calcd forC₂₅H₂₂N₂O₅ (M+Na): 453.1421. Found: 453.1425.

Part F—Preparation ofN-({4-[2-(aminooxy)ethyl]phenyl}methyl)(phenylmethoxy)carboxamide,hydrochloric acid salt

A solution of the product of Part 13E (0.341 g, 0.792 mmol) in 9:1CHCl₃/MeOH (8.00 mL) was treated with hydrazine hydrate (0.190 mL, 3.92mmol) in one portion at 22° C. Within 5 min a white precipitate formed;after 1 h the reaction was complete. The suspension was filtered througha plug of silica (25 g) then eluted with 9:1 CH₂Cl₂/MeOH (750 mL) andconcentrated in vacuo to a white solid. The solid was triturated withEt₂O then removed by filtration through a scintered glass funnel. Thefiltrate was further treated with HCl (0.8 mmol; 0.2 mL of a 4 Msolution in dioxane) and the resulting precipitate collected, washedwith Et₂O (10×5 mL) and dried to constant weight in vacuo (0.220 g,0.653; 82.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ 10.94 (2H, brs), 7.78 (1H,brt, J=5.8 Hz), 7.37-7.28 (5H, m), 7.20 (2H, AB, J_(AB)=8.4 Hz), 7.18(2H, AB, J_(AB)=8.4 Hz), 5.03 (2H, s), 4.20 (2H, t, J=6.6 Hz), 4.16 (2H,brd, J=6.0 Hz), 2.90 (2H, t, J=6.5 Hz). HRMS calcd for C₁₇H₂₀N₂O₃ (M+H):301.1547. Found: 301.1550.

Part G—Preparation of(2R)—N-{2-[4-(aminomethyl)phenyl]ethoxy}-2-[(tert-butoxy)carbonyl-amino]-4-methylpentanamide,trifluoroacetic acid salt

A solution of Boc-DLeu-OH (49.0 mg, 0.197 mmol) in DMF (1.00 mL) wassuccessively treated with HOBt (30.0 mg, 0.196 mmol), i-Pr₂NEt (51 μL,0.293 mmol) and HBTU (75.0 mg, 0.198 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 13F (55.0 mg, 0.163 mmol)in one portion. The resulting solution was stirred 0.5 h then dilutedwith EtOAc (25 mL) and transferred to a separatory funnel. The EtOAcsolution was successively washed with 0.1 M citric acid (3×30 mL) andsaturated aqueous solutions of NaHCO₃ (3×30 mL) and NaCl (30 mL) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The crude hydroxamate ester (0.163 mmol theoretical) was dissolved inMeOH (1.00 mL) and treated with 10% Pd on carbon (17.4 mg, 16.3 μmol; 10mol %) in one portion at 22° C. The resulting suspension was spargedwith 1 atm H₂, and maintained 1 h. After purging the vessel with N₂, thesuspension was filtered through a 0.45 μm Acrodisk then concentrated invacuo. The residue was redissolved in 1:1 MeCN/H₂O (3.00 mL) thendirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 15-45% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 20 min was collected andlyophilized to a white powder (61.2 mg, 0.124 mmol; 75.9%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.15 (1H, brs), 8.12 (2H, brs), 7.36 (2H, AB,J_(AB)=8.1 Hz), 7.33 (2H, AB, J_(AB)=8.1 Hz), 6.91 (1H, brd, J=7.6 Hz),3.99 (2H, brs), 3.98-3.89 (2H, m), 3.82-3.78 (1H, m), 2.87 (2H, brt,J=6.2 Hz), 1.58-1.52 (1H, m), 1.46-1.40 (1H, m), 1.36 (9H, s), 1.36-1.31(1H, m), 0.87 (3H, d, J=6.5 Hz), 0.84 (3H, d, J=6.5 Hz). HRMS calcd forC₂₀H₃₃N₃O₄ (M+H): 380.2544. Found: 380.2548.

Part H—Preparation of-{[2-({[N-({4-[2-((2R)-2-amino-4-methylpentanoylaminooxy)ethyl]-phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)-ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (51.1 mg, 82.7 μmol), HOBt (12.7 mg, 82.9 μmol) and the product ofPart 13G (34.0 mg, 68.9 μmol) in dry DMF (2.00 mL) was successivelytreated with i-Pr₂NEt (21 μL, 120 μmol) and HBTU (31.4 mg, 82.8 μmol) at22° C. The resulting solution was stirred 1 h then diluted with EtOAc(15 mL) and successively washed with 0.1 M citric acid (3×10 mL) andsaturated aqueous solutions of NaHCO₃ (3×10 mL) and NaCl (10 mL) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The protected conjugate (68.9 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (2 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved under a stream of N₂ and the white solid residue redissolved inH₂O containing 0.1% TFA and 10% MeCN (3.00 mL) then directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 2-24% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 19 min was collected and lyophilized toa white powder (54.4 mg, 54.5 μmol; 79.2%). ¹H NMR (DMSO-d₆, 600 MHz): δ11.80 (1H, brs), 8.92 (1H, brt, J=5.7 Hz), 8.28 (2H, brs), 7.24 (2H, AB,J_(AB)=8.4 Hz), 7.21 (2H, AB, J_(AB)=8.4 Hz), 4.32 (2H, brd, J=5.6 Hz),4.23 (2H, s), 4.00 (2H, ABXY, J_(AB)=9.6 Hz, J_(AX)=J_(AY)=7.0 Hz,J_(BX)=J_(BY)=6.7 Hz), 3.66 (1H, brs), 3.50 (8H, s), 3.38 (4H, brt,J=5.7 Hz), 3.05 (4H, brt, J=5.7 Hz), 2.87 (2H, ABXY, J_(AX)=J_(AY)=7.0Hz, J_(BX)=J_(BY)=6.7 Hz), 1.60-1.50 (3H, m), 0.90 (3H, d, J=6.1 Hz),0.88 (3H, d, J=6.1 Hz). ¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 165.5,164.6, 158.0 (q, J=31.8 Hz), 136.8, 136.2, 128.8, 127.4, 116.9 (q, J=299Hz), 75.9, 54.3, 53.8, 52.2, 48.9, 48.6, 42.1, 40.0, 33.4, 23.8, 22.2,22.0. HRMS calcd for C₂₉H₄₆N₆O₁₁ (M+H): 655.3297. Found: 655.3291.

Example 142-{[2-({[N-({4-[2-((2R)-2-amino-4-phenylbutanoylaminooxy)ethyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-{2-[4-(aminomethyl)phenyl]ethoxy}-2-[(tert-butoxy)carbonyl-amino]-4-phenylbutanamide,trifluoroacetic acid salt

A solution of Boc-DHfe-OH (55.0 mg, 0.197 mmol) in DMF (1.00 mL) wassuccessively treated with HOBt (30.0 mg, 0.196 mmol), i-Pr₂NEt (51 μL,0.293 mmol) and HBTU (75.0 mg, 0.198 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 13F (55.0 mg, 0.163 mmol)in one portion. The resulting solution was stirred 0.5 h then dilutedwith EtOAc (25 mL) and transferred to a separatory funnel. The EtOAcsolution was successively washed with 0.1 M citric acid (3×30 mL) andsaturated aqueous solutions of NaHCO₃ (3×30 mL) and NaCl (30 mL) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The crude hydroxamate ester (0.163 mmol theoretical) was dissolved inMeOH (1.00 mL) and treated with 10% Pd on carbon (17.4 mg, 16.3 μmol; 10mol %) in one portion at 22° C. The resulting suspension was spargedwith 1 atm H₂, and maintained 1 h. After purging the vessel with N₂, thesuspension was filtered through a 0.45 μm Acrodisk then concentrated invacuo. The residue was redissolved in 1:1 MeCN/H₂O (3.00 mL) thendirectly purified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm)using a 1%/min gradient from 25-51% MeCN containing 0.1% TFA and 10% H₂Oat 20 mL/min. The main product peak eluting at 17 min was collected andlyophilized to a white powder (25.0 mg, 46.2 μmol; 28.3%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.14 (1H, brs), 8.11 (2H, brs), 7.36 (2H, AB,J_(AB)=8.2 Hz), 7.33 (2H, AB, J_(AB)=8.2 Hz), 7.26 (2H, dd, J=7.7, 7.4Hz), 7.18-7.16 (3H, m), 7.08 (1H, brd, J=7.4 Hz), 3.98 (2H, s),3.97-3.91 (2H, m), 3.75 (1H, brs), 2.88 (2H, brdd, J=6.6, 6.1 Hz),2.63-2.58 (1H, m), 2.53-2.47 (1H, m), 1.82-1.78 (2H, m), 1.38 (9H, s).HRMS calcd for C₂₄H₃₃N₃O₄ (M+H): 428.2544. Found: 428.2542.

Part B—Preparation of2-{[2-({[N-({4-[2-((2R)-2-amino-4-phenylbutanoylaminooxy)ethyl]-phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)-ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (31.5 mg, 51.0 μmol), HOBt (7.8 mg, 51 μmol) and the product ofPart 14A (23.0 mg, 42.5 μmol) in dry DMF (2.00 mL) was successivelytreated with i-Pr₂NEt (13 μL, 75 μmol) and HBTU (19.3 mg, 50.9 μmol) at22° C. The resulting solution was stirred 1 h then diluted with EtOAc(15 mL) and successively washed with 0.1 M citric acid (3×10 mL) andsaturated aqueous solutions of NaHCO₃ (3×10 mL) and NaCl (10 mL) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The protected conjugate (42.5 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (2 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved under a stream of N₂ and the white solid residue redissolved inH₂O containing 0.1% TFA and 10% MeCN (3.00 mL) then directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 7-29% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 16 min was collected and lyophilized toa white powder (13.3 mg, 12.7 μmol; 30.0%). ¹H NMR (DMSO-d₆, 600 MHz): δ11.78 (1H, brs), 8.90 (1H, brt, J=5.6 Hz), 8.35 (2H, brs), 7.30 (2H, dd,J=7.6, 7.6 Hz), 7.25 (2H, AB, J_(AB)=7.9 Hz), 7.21 (2H, AB, J_(AB)=7.9Hz), 7.18 (2H, d, J=7.3 Hz), 7.21-7.17 (1H, m), 4.31 (2H, brd, J=5.2Hz), 4.23 (2H, s), 4.03 (2H, ABXY, J_(AB)=9.7 Hz, J_(AX)=J_(AY)=7.0 Hz,J_(BX)=J_(BY)=6.7 Hz), 3.68 (1H, brs), 3.49 (8H, s), 3.38 (4H, brt,J=5.5 Hz), 3.04 (4H, brt, J=5.8 Hz), 2.89 (2H, ABXY,J_(AX)=J_(BX)=J_(AY)=J_(BY)=6.7 Hz), 2.59 (2H, dd, J=8.5, 8.2 Hz),2.03-1.93 (2H, m). ¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 165.2, 164.6,157.9 (q, J=31.8 Hz), 140.2, 136.8, 136.2, 128.8, 128.5, 128.0, 127.4,126.2, 116.9 (q, J=299 Hz), 76.0, 54.3, 53.8, 52.2, 50.3, 48.6, 42.1,33.4, 32.7, 30.4. HRMS calcd for C₃₃H₄₆N₆O₁₁ (M+H): 703.3297. Found:703.3289.

Example 152-{[2-({[N-({4-[2-((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)ethyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)—N-{2-[4-(aminomethyl)phenyl]ethoxy}-2-[(tert-butoxy)carbonyl-amino]-3-(2-naphthyl)propanamide,trifluoroacetic acid salt

A solution of Boc-DNal-OH (62.0 mg, 0.197 mmol) in DMF (1.00 mL) wassuccessively treated with HOBt (30.0 mg, 0.196 mmol), i-Pr₂NEt (51 μL,0.293 mmol) and HBTU (75.0 mg, 0.198 mmol) at 22° C. After 0.25 h, thesolution was treated with the product of Part 13F (55.0 mg, 0.163 mmol)in one portion. The resulting solution was stirred 0.5 h then dilutedwith EtOAc (25 mL) and transferred to a separatory funnel. The EtOAcsolution was successively washed with 0.1 M citric acid (3×30 mL) andsaturated aqueous solutions of NaHCO₃ (3×30 mL) and NaCl (30 mL) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The crude hydroxamate ester (0.163 mmol theoretical) was dissolved inMeOH (1.00 mL) and treated with 10% Pd on carbon (17.4 mg, 16.3 μmol; 10mol %) in one portion at 22° C. The resulting suspension was spargedwith 1 atm H₂, and maintained 2 h; an additional 0.2 equiv Pd was addedafter 1 h to ensure complete conversion. After purging the vessel withN₂, the suspension was filtered through a 0.45 μm Acrodisk thenconcentrated in vacuo. The residue was redissolved in 1:1 MeCN/H₂O (3.00mL) then directly purified by HPLC on a Phenomenex Luna C18 column(21.2×250 mm) using a 1%/min gradient from 25-51% MeCN containing 0.1%TFA and 10% H₂O at 20 mL/min. The main product peak eluting at 18 minwas collected and lyophilized to a white powder (60.8 mg, 0.105 mmol;64.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.14 (1H, brs), 8.11 (2H, brs),7.85 (1H, d, J=7.3 Hz), 7.82 (1H, d, J=8.4 Hz), 7.80 (1H, d, J=7.6 Hz),7.71 (1H, s), 7.48-7.44 (2H, m), 7.41 (1H, d, J=8.1 Hz), 7.33 (2H, AB,J_(AB)=7.8 Hz), 7.21 (2H, AB, J_(AB)=7.3 Hz), 7.14 (1H, brd, J=7.8 Hz),4.13-4.09 (1H, m), 3.98 (2H, s), 3.86-3.82 (1H, m), 3.76-3.72 (1H, m),3.40 (1H, ABXY, J_(AB)=13.3 Hz, J_(AX)=J_(AY)=6.5 Hz), 2.97 (1H, ABXY,J_(AB)=13.5 Hz, J_(BX)=J_(BY)=8.7 Hz), 2.71 (2H, brs), 1.29 (9H, s).HRMS calcd for C₂₇H₃₃N₃O₄ (M+H): 464.2544. Found: 464.2538.

Part B—Preparation of2-{[2-({[N-({4-[2-((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)-ethyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)-ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (46.2 mg, 74.8 μmol), HOBt (11.5 mg, 75.1 μmol) and the product ofPart 15A (36.0 mg, 62.3 μmol) in dry DMF (2.00 mL) was successivelytreated with i-Pr₂NEt (19 μL, 110 μmol) and HBTU (28.4 mg, 74.9 μmol) at22° C. The resulting solution was stirred 1 h then diluted with EtOAc(15 mL) and successively washed with 0.1 M citric acid (3×10 mL) andsaturated aqueous solutions of NaHCO₃ (3×10 mL) and NaCl (10 mL) thendried over MgSO₄, filtered and concentrated in vacuo to a colorless oilwhich was used without further purification in the subsequentdeprotection step.

The protected conjugate (62.3 μmol theoretical) was dissolved in dioxane(0.500 mL) then successively treated with H₂O (2 μL) and HCl (2.00 mmol;0.500 mL of a 4 M solution in dioxane) at 22° C. The resulting paleyellow solution was stirred 18 h, during which time a heavy whiteprecipitate formed. Upon complete deprotection, the volatiles wereremoved under a stream of N₂ and the white solid residue redissolved inH₂O containing 0.1% TFA and 10% MeCN (3.00 mL) then directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 12-32% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 20 min was collected and lyophilized toa white powder (36.5 mg, 33.8 μmol; 54.2%). ¹H NMR (DMSO-d₆, 600 MHz): δ11.55 (1H, brs), 8.90 (1H, brt, J=5.7 Hz), 8.47 (2H, brs), 7.90-7.87(2H, m), 7.84-7.81 (1H, m), 7.72 (1H, s), 7.50-7.46 (2H, m), 7.38 (1H,brd, J=8.3 Hz), 7.14 (2H, AB, J_(AB)=8.0 Hz), 7.00 (2H, AB, J_(AB)=8.0Hz), 4.29 (2H, brd, J=5.5 Hz), 4.23 (2H, s), 3.90 (1H, brs), 3.79 (1H,ABXY, J_(AB)=10.0 Hz, J_(AX)=J_(AY)=7.0 Hz), 3.64 (1H, ABXY, J_(AB)=10.0Hz, J_(BX)=J_(BY)=6.8 Hz), 3.50 (8H, s), 3.38 (4H, brt, J=5.6 Hz), 3.22(1H, ABX, J_(AB)=13.2 Hz, J_(AX)=5.6 Hz), 3.16 (1H, ABX, J_(AB)=13.2 Hz,J_(BX)=8.6 Hz), 3.05 (4H, brt, J=5.7 Hz), 2.56 (2H, ABXY,J_(AX)=J_(BX)=J_(AY)=J_(BY)=6.9 Hz). ¹³C NMR (DMSO-d₆, 151 MHz): δ172.7, 164.6, 164.4, 158.0 (q, J=32.9 Hz), 136.6, 136.1, 132.9, 132.3,132.2, 128.7, 128.1, 127.5, 127.4, 127.4, 127.3, 126.2, 125.9, 116.7 (q,J=297 Hz), 75.7, 54.3, 53.8, 52.2, 51.5, 48.6, 42.1, 37.0, 33.1. HRMScalcd for C₃₆H₄₆N₆O₁₁ (M+H): 739.3297. Found: 739.32.

Example 162-{7-[(N-{[4-({[(1R)-1-(N-methoxycarbamoyl)-3-phenylpropyl]amino}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl}aceticacid, trifluoroacetic acid salt

Part A—Preparation of (2R)-2-Amino-N-methoxy-4-phenylbutanamide

A solution of Boc-DHfe-OH (1.40 g, 5.00 mmol) and HOBt (0.919 g, 6.00mmol) in dry DMF (25.0 mL) was successively treated with i-Pr₂NEt (2.09mL, 12.0 mmol) and HBTU (2.28 g, 6.00 mmol) then stirred 0.25 h at 22°C. The resulting solution was treated with MeONH₂.HCl (0.501 g, 6.00mmol) in one portion, maintained 0.5 h then partitioned between EtOAcand 0.1 M HCl (50 ml each) with transfer to a separatory funnel. Thelayers separated and the aqueous layer washed with EtOAc (2×50 mL). Thecombined EtOAc washes were successively washed with 0.1 M HCl, 0.1 MNaOH and saturated aqueous NaCl (3×50 mL each) then dried over MgSO₄,filtered and concentrated in vacuo to a white solid (R_(f)=0.2 in 1:1hexanes/EtOAc).

The crude methyl hydroxamate was redissolved in dioxane (75.0 mL) thensuccessively treated with Et₃SiH (799 μL, 5.00 mmol) and HCl (0.100 mol;25.0 mL of a 4.0 M solution in dioxane) at 22° C. The resulting solutionwas stirred 12.5 h then neutralized with 1.0 M NaOH (100 mL), dilutedwith EtOAc (100 mL) and transferred to a separatory funnel. The layersseparated and the aqueous layer exhaustively washed with EtOAc (6×50mL). The combined EtOAc layers were dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil that was purified bychromatography on silica (40×210 mm) using 9:1 CH₂Cl₂/MeOH containing1.0% Et₃N(R_(f)=0.1 in 9:1 CH₂Cl₂/MeOH). The main product eluted between300-420 mL, was collected and concentrated to afford an amorphous whitepowder (0.659 g, 3.16 mmol; 63.3%). ¹H NMR (DMSO-d₆, 300 MHz): δ7.30-7.14 (5H, m), 3.58 (3H, s), 3.02 (1H, dd, J=7.5, 6.0 Hz), 2.69-2.50(2H, m), 1.80 (1H, dddd, J=13.2, 10.1, 6.2, 6.2 Hz), 1.64 (1H, dddd,J=13.4, 10.0, 7.8, 5.8 Hz). ¹³C NMR (DMSO-d₆, 75 MHz): δ 171.6, 141.8,128.2, 128.2, 125.7, 63.0, 52.4, 36.8, 31.4. HRMS calcd for C₁₁H₁₆N₂O₂(M+H): 209.1285. Found: 209.1288.

Part B—Preparation ofN-[(4-formylphenyl)methyl]prop-2-enyloxycarboxamide

A solution of the product of Part 1B (2.21 g, 10.0 mmol) in dry CH₂Cl₂(50.0 mL) was treated with Dess-Martin periodinane (5.09 g, 12.0 mmol)in one portion at 22° C. Within one min, rapid dissolution of theoxidant was observed; leading to gentle reflux of the reaction mixture.After 5 min, complete oxidation was observed and the resultingsuspension diluted with Et₂O (50 mL). The solids were removed byfiltration through a pad of Celite and the filter cake exhaustivelywashed with Et₂O; final filtrate volume of 500 mL. The combinedfiltrates were concentrated in vacuo to a pale yellow oil then purifiedby chromatography on silica (40×265 mm) using a step gradient from3:2→2:3 hexanes/EtOAc (R_(f)=0.5 in 1:1 hexanes/EtOAc) to afford thepure product as a colorless oil (2.15 g, 9.81 mmol; 98.1%) ¹H NMR(CDCl₃, 600 MHz): δ 10.01 (1H, s), 7.86 (2H, AB, J_(AB)=8.1 Hz), 7.47(2H, AB, J_(AB)=7.9 Hz), 5.59 (1H, ddt, J=16.9, 10.7, 5.6 Hz) 5.33 (1H,d, J=17.0 Hz), 5.24 (1H, d, J=10.4 Hz), 5.22 (1H, brs), 4.62 (2H, dt,J=5.7, 1.5 Hz), 4.47 (2H, d, J=6.1 Hz). ¹³C NMR (CDCl₃, 151 MHz): δ191.8, 156.3, 145.5, 135.7, 132.6, 130.1, 127.8, 117.9, 65.9, 44.7. HRMScalcd for C₁₂H₁₃NO₃ (M+H): 220.0968. Found: 220.0967.

Part C—Preparation of(2R)—N-methoxy-4-phenyl-2-[({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methyl)amino]butanamide,hydrochloric acid salt

A solution of the product of Parts 16A (0.177 g, 0.850 mmol) and 16B(0.186 g, 0.850 mmol) in dry MeOH (8.50 mL) was cooled to 0° C. thentreated with NaCNBH₃ (0.160 g, 2.55 mmol) in one portion. After 1 h,glacial AcOH (0.048 mL, 0.850 mmol) was added to the reaction mixture; adramatic increase in conversion was observed. The AcOH treatment processwas then repeated two additional times during the next 2 h, maintaininga 1 h interval between each equivalent. After 4 h total reaction time,the resulting solution was partitioned between EtOAc and saturatedaqueous NaHCO₃ (50 mL each) with transfer to a separatory funnel. Thelayers separated and the aqueous layer washed with EtOAc (2×50 mL). Thecombined EtOAc layers were then dried over MgSO₄, filtered andconcentrated in vacuo to a pale yellow oil. Purification bychromatography on silica (40×260 mm) using 98:2 EtOAc/MeOH afforded thepure product as a colorless oil. The oil was then redissolved in dryEt₂O (100 mL) and treated with HCl (4.00 mmol; 1.00 mL of a 4.0 Msolution in dioxane) at 22° C. The resulting suspension was filteredthrough a scintered glass funnel of medium porosity and the collectedsolids exhaustively washed with Et₂O then dried in vacuo to an amorphouswhite powder (0.253 g, 0.564 mmol; 66.3%). ¹H NMR (DMSO-d₆, 600 MHz): δ12.20 (1H, s), 10.16 (1H, brs), 9.53 (1H, brs), 7.83 (1H, brt, J=6.1Hz), 7.51 (2H, AB, J_(AB)=8.1 Hz), 7.31-7.27 (4H, m), 7.22-7.18 (3H, m),5.91 (1H, ddt, J=17.1, 10.6, 5.4 Hz), 5.28 (1H, dq, J=17.2, 1.7 Hz),5.18 (1H, dq, J=10.5, 1.5 Hz), 4.49 (2H, dt, J=5.4, 1.5 Hz), 4.20 (2H,d, J=6.2 Hz), 4.13-3.99 (2H, m), 3.68 (3H, s), 3.47 (1H, brs), 2.63 (2H,ABXY, J_(AB)=13.6 Hz, J_(AX)=J_(BX)=10.9 Hz, J_(AY)=J_(BY)=5.9 Hz)2.24-2.16 (1H, m), 2.10 (1H, dddd, J=13.5, 10.8, 8.6, 6.3 Hz). ¹³C NMR(DMSO-d₆, 151 MHz): δ 163.5, 156.2, 140.8, 140.2, 133.7, 130.3, 129.7,128.4, 128.1, 127.0, 126.2, 116.9, 64.4, 63.6, 56.7, 48.6, 43.4, 31.3,30.4.

Part D—Preparation of2-{7-[(N-{[4-({[(1R)-1-(N-methoxycarbamoyl)-3-phenylpropyl]amino}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl}aceticacid, trifluoroacetic acid salt

The product of Part 16C (112 mg, 0.250 mmol) was dissolved in 2:1MeCN/H₂O (5.00 mL) and successively treated with 14.2 mg TPPTS (25.0μmol; 10 mol %), Et₂NH (129 μL, 1.25 mmol) and 2.8 mg Pd(OAc)₂ (12.5μmol; 5 mol %) at 22° C. Complete deprotection was observed within 0.25h. The resulting amber solution was then lyophilized to remove allvolatile components.

The solids thus obtained were redissolved in DMF and successivelytreated with HOBt (45.9 mg, 0.300 mmol),2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}-cyclododecyl)aceticacid (172 mg, 0.300 mmol), i-Pr₂NEt (105 μL, 0.600 mmol) and HBTU (114mg, 0.300 mmol) at 22° C. After 0.25 h, complete acylation was observed;only trace amounts of regioisomeric and dimeric products formed. Theresulting solution was partitioned between EtOAc and H₂O (50 mL each)with transfer to a separatory funnel. The layers separated and theaqueous layer washed with EtOAc (2×50 mL). The EtOAc solution wasfurther washed with 0.1 M NaOH (3×50 mL) and saturated aqueous NaCl(3×50 mL each), then dried over MgSO₄, filtered and concentrated invacuo to a pale yellow oil that was used without further purification inthe subsequent deprotection step.

The protected conjugate (0.250 mmol theoretical) was dissolved indioxane (2.50 mL) then successively treated with H₂O (23 μL) and HCl(10.0 mmol; 2.50 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 17 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O containing 0.1% TFA (8.00 mL) then partially purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 2%/mingradient from 0-60% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 22 min was collected and lyophilized toa white powder. Final purification was performed using the identicalcolumn and method. The main product peak was collected and lyophilizedto a white powder (99.0 mg, 93.8 μmol; 37.5%). ¹H NMR (methanol-d₄, 600MHz): δ 7.44 (2H, AB, J_(AB)=8.3 Hz), 7.41 (2H, AB, J_(AB)=8.3 Hz),7.31-7.27 (2H, m), 7.20 (3H, m), 4.40 (2H, s), 4.16 (2H, ABq,J_(AB)=13.0 Hz), 3.84-3.74 (9H, brm), 3.78 (3H, s), 3.35 (8H, brs), 3.25(8H, brs), 2.72-2.62 (2H, m), 2.24-2.13 (2H, m). ¹³C NMR (methanol-d₄,151 MHz): δ 165.7, 163.0 (q, J_(CF)=34.6 Hz), 142.0, 141.1, 131.7,130.8, 129.9, 129.8, 129.4, 127.8, 118.3 (q, J_(CF)=293 Hz), 65.0, 59.1,56.2, 55.6 (br), 55.1 (br), 51.5 (br), 51.1, 51.0 (br) 44.0, 33.5, 32.2.HRMS calcd for C₃₅H₅₁N₇O₉ (M+H): 714.3821. Found: 714.3819.

Example 172-(7-{[N-({4-[({(1R)-3-phenyl-1-[N-(phenylmethoxy)carbamoyl]propyl}amino)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

Part A—Preparation of (2R)-2-Amino-4-phenyl-N-(phenylmethoxy)butanamide

A solution of Boc-DHfe-OH (1.40 g, 5.00 mmol) and HOBt (0.919 g, 6.00mmol) in dry DMF (25.0 mL) was successively treated with i-Pr₂NEt (2.09mL, 12.0 mmol) and HBTU (2.28 g, 6.00 mmol) then stirred 0.25 h at 22°C. The resulting solution was treated with BnONH₂.HCl (0.958 g, 6.00mmol) in one portion, maintained 0.5 h then partitioned between EtOAcand 0.1 M HCl (50 ml each) with transfer to a separatory funnel. Thelayers separated and the aqueous layer washed with EtOAc (2×50 mL). Thecombined EtOAc washes were successively washed with 0.1 M HCl, 0.1 MNaOH and saturated aqueous NaCl (3×50 mL each) then dried over MgSO₄,filtered and concentrated in vacuo to a white solid (R_(f)=0.5 in 1:1hexanes/EtOAc).

The crude benzyl hydroxamate was redissolved in dioxane (75.0 mL) thensuccessively treated with Et₃SiH (799 μL, 5.00 mmol) and HCl (0.100 mol;25.0 mL of a 4.0 M solution in dioxane) at 22° C. The resulting solutionwas stirred 12.5 h then neutralized with 1.0 M NaOH (100 mL), dilutedwith EtOAc (100 mL) and transferred to a separatory funnel. The layersseparated and the aqueous layer exhaustively washed with EtOAc (3×50mL). The combined EtOAc layers were dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil that was purified bychromatography on silica (50×170 mm) using 9:1 CH₂Cl₂/MeOH containing1.0% Et₃N (R_(f)=0.3 in 9:1 CH₂Cl₂/MeOH). The main product elutedbetween 320-480 mL, was collected and concentrated to afford anamorphous white powder (1.19 g, 4.18 mmol; 83.7%). ¹H NMR (DMSO-d₆, 600MHz): δ 7.41-7.32 (5H, m), 7.28-7.25 (2H, m), 7.17-7.15 (3H, m), 4.81(2H, s), 3.02 (1H, dd, J=7.3, 6.1 Hz), 2.55 (2H, ABXY, J_(AB)=13.7 Hz,J_(AX)=J_(BX)=10.3 Hz, J_(AY)=5.6 Hz, J_(BY)=6.2 Hz), 1.78 (1H, ddt,J=13.2, 10.2, 6.1 Hz), 1.63 (1H, dddd, J=13.1, 10.2, 7.5, 5.6 Hz). ¹³CNMR (DMSO-d₆, 151 MHz): δ 171.9, 141.8, 136.1, 128.7, 128.2, 128.1,125.6, 76.6, 52.4, 36.9, 31.4. HRMS calcd for C₁₇H₂₀N₂O₂ (M+H):285.1598. Found: 285.1596.

Part B—Preparation of(2R)-4-Phenyl-N-(phenylmethoxy)-2-[({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methyl)amino]butanamide,hydrochloric acid salt

A solution of the product of Parts 17A (0.270 g, 0.950 mmol) and 16B(0.208 g, 0.950 mmol) in dry MeOH (8.50 mL) was cooled to 0° C. thentreated with NaCNBH₃ (0.179 g, 2.85 mmol) in one portion. After 1 h,glacial AcOH (0.054 mL, 0.950 mmol) was added to the reaction mixture; adramatic increase in conversion was observed. The AcOH treatment processwas then repeated two additional times during the next 2 h, maintaininga 1 h interval between each equivalent. After 4 h total reaction time,the resulting solution was partitioned between EtOAc and saturatedaqueous NaHCO₃ (50 mL each) with transfer to a separatory funnel. Thelayers separated and the aqueous layer washed with EtOAc (2×50 mL). Thecombined EtOAc layers were then dried over MgSO₄, filtered andconcentrated in vacuo to a pale yellow oil. Purification bychromatography on silica (40×250 mm) using 98:2 EtOAc/MeOH afforded thepure product as a colorless oil. The oil was then redissolved in dryEt₂O (100 mL) and treated with HCl (4.00 mmol; 1.00 mL of a 4.0 Msolution in dioxane) at 22° C. The resulting suspension was filteredthrough a scintered glass funnel of medium porosity and the collectedsolids exhaustively washed with Et₂O then dried in vacuo to an amorphouswhite powder (0.330 g, 0.629 mmol; 66.2%). ¹H NMR (DMSO-d₆, 600 MHz): δ12.11 (1H, s), 10.14 (1H, brs), 9.52 (1H, brs), 7.83 (1H, brt, J=6.1Hz), 7.49-7.44 (4H, m), 7.40-7.37 (2H, m), 7.36-7.33 (1H, m), 7.30-7.27(4H, m), 7.21-7.18 (1H, m), 7.14-7.12 (2H, m), 5.92 (1H, ddt, J=17.2,10.6, 5.4 Hz), 5.29 (1H, dq, J=17.2, 1.7 Hz), 5.18 (1H, dq, J=10.5, 1.5Hz), 4.94 (2H, s), 4.49 (2H, dt, J=5.4, 1.5 Hz), 4.20 (2H, d, J=6.2 Hz),4.02-3.90 (2H, m), 3.42 (1H, brs), 2.50 (2H, ABXY, J_(AB)=13.8 Hz,J_(AX)=J_(BX)=10.9 Hz, J_(AY)=J_(BY)=5.8 Hz), 2.17-2.11 (1H, m), 2.05(1H, dddd, J=13.4, 11.1, 8.7, 6.0 Hz). ¹³C NMR (DMSO-d₆, 151 MHz): δ163.7, 156.2, 140.8, 140.2, 135.6, 133.7, 130.3, 129.6, 128.8, 128.4(2),128.3, 128.1, 127.0, 126.1, 116.9, 77.1, 64.4, 56.7, 48.5, 43.4, 31.3,30.3.

Part C—Preparation of2-(7-{[N-({4-[({(1R)-3-phenyl-1-[N-(phenylmethoxy)carbamoyl]propyl}amino)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

The product of Part 17B (131 mg, 0.250 mmol) was dissolved in 2:1MeCN/H₂O (5.00 mL) and successively treated with 14.2 mg TPPTS (25.0μmol; 10 mol %), Et₂NH (129 μL, 1.25 mmol) and 2.8 mg Pd(OAc)₂ (12.5μmol; 5 mol %) at 22° C. Complete deprotection was observed within 0.25h. The resulting amber solution was then lyophilized to remove allvolatile components.

The solids thus obtained were redissolved in DMF and successivelytreated with HOBt (45.9 mg, 0.300 mmol),2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}-cyclododecyl)aceticacid (172 mg, 0.300 mmol), i-Pr₂NEt (105 μL, 0.600 mmol) and HBTU (114mg, 0.300 mmol) at 22° C. After 0.25 h, complete acylation was observed;only trace amounts of regioisomeric and dimeric products formed. Theresulting solution was partitioned between EtOAc and H₂O (50 mL each)with transfer to a separatory funnel. The layers separated and theaqueous layer washed with EtOAc (2×50 mL). The EtOAc solution wasfurther washed with 0.1 M NaOH (3×50 mL) and saturated aqueous NaCl(3×50 mL each), then dried over MgSO₄, filtered and concentrated invacuo to a pale yellow oil that was used without further purification inthe subsequent deprotection step.

The protected conjugate (0.250 mmol theoretical) was dissolved indioxane (2.50 mL) then successively treated with H₂O (23 μL) and HCl(10.0 mmol; 2.50 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 17 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O containing 0.1% TFA (8.00 mL) then partially purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 2%/mingradient from 0-60% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 21 min was collected and lyophilized toa white powder. Final purification was performed using the identicalcolumn and method. The main product peak was collected and lyophilizedto a white powder (0.110 g, 97.3 μmol; 38.9%). ¹H NMR (methanol-d₄, 600MHz): δ 7.50-7.48 (2H, m), 7.42-7.35 (6H, m), 7.34-7.30 (1H, m),7.28-7.24 (2H, m), 7.21-7.17 (1H, m), 7.12-7.09 (2H, m), 4.98 (2H, ABq,J_(AB)=11.6 Hz), 4.20 (2H, ABq, J_(AB)=15.4 Hz), 3.99 (2H, ABq,J_(AB)=12.9 Hz), 3.84 (7H, brs), 3.68 (1H, dd, J=8.5, 5.1 Hz), 3.33 (8H,brs), 3.28 (8H, brs), 2.63 (2H, ABXY, J_(AB)=13.8 Hz, J_(AX)=J_(BX)=10.0Hz, J_(AY)=J_(BY)=7.1 Hz) 2.17-2.03 (2H, m). ¹³C NMR (methanol-d₄, 151MHz): δ 165.6, 162.93 (q, J_(CF)=34.7 Hz), 141.9, 141.1, 136.9, 131.7,130.8, 130.5, 130.1, 129.8, 129.8, 129.7, 129.4, 127.7, 118.3 (q,J_(CF)=293 Hz), 79.3, 59.3, 56.1, 55.3 (br), 55.0 (br), 51.4 (br), 51.1,44.0, 33.5, 32.1. HRMS calcd for C₄₁H₅₅N₇O₉ (M+H): 790.4134. Found:790.4129.

Example 182-(4-{[N-({4-[(2R)-2-amino-2-(N-methoxycarbamoyl)ethyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-7,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)-3-[4-(Aminomethyl)phenyl]-2-[(tert-butoxy)carbonyl-amino]propanoicacid, trifluoroacetic acid salt

(2R)-2-[(tert-Butoxy)carbonylamino]-3-(4-cyanophenyl)propanoic acid(0.581 g, 2.00 mmol) was dissolved in a solution of 28% aqueous. NH₃ inMeOH (1:2 v/v; 24 mL), then carefully treated with 0.6 g Raney Ni 2800under a N₂ atmosphere. Using a Parr apparatus, the headspace of the 250mL reaction vessel was repeatedly sparged with H₂, then pressurized to50 psi and shaken 4 h at 22° C. Upon complete conversion, the headspacewas evacuated then repeatedly sparged with N₂. The resulting suspensionwas filtered through a pad of Celite and the filter cake (plus reactionvessel) exhaustively washed with small portions of 1:1 MeCN/H₂O; 100 mLfinal wash volume. The filtrate was neutralized with glacial AcOH, thendiluted with H₂O (100 mL) and partially concentrated in vacuo; 175 mLfinal volume. Lyophilzation of this solution provided the crude productas a white solid suitable for use in the subsequent coupling step. Ifdesired, the crude material may be purified by HPLC on a Phenomenex LunaC18 column (21.2×250 mm) using a 1%/min gradient from 0-40% MeCNcontaining 0.1% TFA and 10% H₂O at 20 mL/min. The main product peakeluting at 24 min was collected and lyophilized to a whitemicrocrystalline solid. All spectroscopic data of this material wasconsistent with published reports.

Part B—Preparation of2-(4-{[N-({4-[(2R)-2-amino-2-(N-methoxycarbamoyl)ethyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-7,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

A solution of 2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}-cyclododecyl)acetic acid (68.7 mg,0.120 mmol) in dry DMF (1.00 mL) was successively treated with HOBt(18.4 mg, 0.120 mmol) and EDC (22.9 mg, 0.120 mmol) at 22° C. After 0.5h, the solution was treated with the product of Part 18A (40.8 mg, 0.100mmol) and the resulting mixture stirred 0.5 h. The intermediateconjugate thus obtained was once again activated with EDC (22.9 mg,0.120 mmol), then stirred 0.5 h before final treatment with MeONH₂.HCl(10.0 mg, 0.120 mmol). After 1 h, the resulting mixture was diluted withEtOAc (100 mL) then transferred to a separatory funnel and successivelywashed with 0.1 M NaOH and saturated aqueous NaCl (3×25 mL each). TheEtOAc solution was dried over MgSO₄, filtered and concentrated in vacuoto a colorless oil, which was used without further purification in thesubsequent deprotection step.

The protected conjugate (0.120 mmol theoretical) was dissolved indioxane (1.00 mL) then successively treated with H₂O (9 μL) and HCl(4.00 mmol; 1.00 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 14 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O containing 0.1% TFA (8.00 mL) then directly purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 0-30% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 11.5 min was collected and lyophilizedto a white powder (12.8 mg, 13.4 μmol; 13.4%). ¹H NMR (methanol-d₄, 600MHz): δ 7.33 (2H, AB, J_(AB)=8.0 Hz), 7.22 (2H, AB, J_(AB)=8.1 Hz), 4.36(2H, brs), 3.84 (5H, brs), 3.75-3.66 (4H, brm), 3.57 (3H, s), 3.37 (8H,brs), 3.31 (8H, brs), 3.14-3.06 (2H, m). HRMS calcd for C₂₇H₄₃N₇O₉(M+H): 610.3195. Found: 610.3199.

Example 192-[7-({N-[(4-{(2R)-2-amino-2-[N-(phenylmethoxy)carbamoyl]ethyl}phenyl)methyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticacid, trifluoroacetic acid salt

A solution of2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}-cyclododecyl)aceticacid (68.7 mg, 0.120 mmol) in dry DMF (1.00 mL) was successively treatedwith HOBt (18.4 mg, 0.120 mmol) and EDC (22.9 mg, 0.120 mmol) at 22° C.After 0.5 h, the solution was treated with the product of Part 18A (40.8mg, 0.100 mmol) and the resulting mixture stirred 0.5 h. Theintermediate conjugate thus obtained was once again activated with EDC(22.9 mg, 0.120 mmol), then stirred 0.5 h before final treatment withBnONH₂.HCl (19.2 mg, 0.120 mmol). After 1 h, the resulting mixture wasdiluted with EtOAc (100 mL) then transferred to a separatory funnel andsuccessively washed with 0.1 M NaOH and saturated aqueous NaCl (3×25 mLeach). The EtOAc solution was dried over MgSO₄, filtered andconcentrated in vacuo to a colorless oil, which was used without furtherpurification in the subsequent deprotection step.

The protected conjugate (0.120 mmol theoretical) was dissolved indioxane (1.00 mL) then successively treated with H₂O (9 μL) and HCl(4.00 mmol; 1.00 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred 14 h, during which time aheavy white precipitate formed. Upon complete deprotection, thevolatiles were removed under a stream of N₂ and the white solid residueredissolved in H₂O containing 0.1% TFA (8.00 mL) then partially purifiedby HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 0-40% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.The main product peak eluting at 21 min was collected and lyophilized toa white powder. Final purification was performed using an identicalcolumn combined with a 1%/min gradient from 0-50% MeCN containing 0.1%HCO₂H and 10% H₂O at 20 mL/min. The main product peak eluting at 14 minwas collected and lyophilized to a white powder (8.2 mg, 10.0 μmol;10.0%). ¹H NMR (methanol-d₄, 600 MHz): δ 7.39 (2H, AB, J_(AB)=7.7 Hz),7.37-7.28 (5H, m), 7.19 (2H, AB, J_(AB)=8.0 Hz), 4.70 (2H, ABq,J_(AB)=1.0 Hz), 4.41 (2H, ABq, J_(AB)=14.8 Hz), 3.84 (1H, brt, J=6.8Hz), 3.66-3.36 (16H, m), 3.11-2.91 (11H, m). HRMS calcd for C₃₃H₄₇N₇O₉(M+H): 686.3508. Found: 686.3518.

Example 202-{[2-({[N-({4-[((2R)-2-amino-4-methylpentanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation ofN-({4-[(1,3-dioxoisoindolin-2-yloxy)methyl]phenyl}methyl)prop-2-enyloxycarboxamide

A solution of N-hydroxyphthalimide (3.32 g, 20.3 mmol), the product ofPart 1B (3.00 g, 13.6 mmol), and PPh₃ (5.33 g, 20.3 mmol) in dry THF(100 mL) was cooled to 0° C. while stirring under N₂. ADDP (5.13 g, 20.3mmol) was added in one portion and the resulting yellow solution warmedto ambient temperature. The solution was stirred for 23 h then heated to50° C. and maintained 5 h. After cooling to 22° C., the THF removed invacuo and the residue partitioned between Et₂O and saturated aqueousNaHCO₃ (500 mL each). The Et₂O layer was washed with additional NaHCO₃solution (2×500 mL) then dried over Na₂SO₄, filtered and concentrated invacuo to afford the crude product as a yellow solid (7.3 g) that wasused without further purification in the subsequent deprotection step.LRMS: 389.2 (100, M+Na), 367.2 (100), 323.2 (25).

Part B—Preparation ofN-({4-[(aminooxy)methyl]phenyl}methyl)prop-2-enyloxycarboxamide

The product of Part 20A (1 g) was dissolved in MeOH (40.0 mL) andhydrazine hydrate (105 mg, 3.3 mmol) added in one portion at 22° C. Themixture was heated to reflux, maintained 0.5 h then cooled to 0° C.using an ice-water bath and maintained 2 h. The white solid precipitatewas removed by filtration through a scintered glass funnel and thefiltrate concentrated to afford the crude product as a pale yellow solid(794 mg) of suitable purity for use in the subsequent coupling reaction.¹H NMR (DMSO-d₆, 600 MHz): δ 8.1 (1H, brs), 7.24 (4H, ABq, J_(AB)=8.0Hz), 6.00 (2H, brs), 5.91 (1H, ddt, J=17.4, 10.2, 5.4 Hz), 5.28 (1H, d,J=17.4 Hz), 5.17 (1H, d, J=10.2 Hz), 4.53 (2H, s), 4.49 (2H, dt, J=5.3,1.5 Hz), 4.18 (2H, d, 6.2 Hz). HRMS calcd for C₁₂H₁₆N₂O₃ (M+H):237.1234. Found: 237.1238.

Part C—Preparation of(2R)-2-[(tert-Butoxy)carbonylamino]-4-methyl-N-({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methoxy)pentanamide

The product of Part 20B (0.200 g, 0.846 mmol) was added to a stirringmixture of Boc-DLeu-OH (254 mg, 1.10 mmol), HOBt (168 mg, 1.10 mmol),HBTU (417 mg, 1.10 mmol), and i-Pr₂NEt (678 μL, 3.89 mmol) in DMF at 22°C. The resulting mixture was stirred overnight then concentrated invacuo and the residue dissolved in EtOAc. The EtOAc solution wassuccessively washed with 0.1 N HCl, 5% aqueous NaHCO₃, and saturatedaqueous NaCl then dried over Na₂SO₄, filtered and concentrated in vacuo.The crude material was purified by HPLC on a Phenomenex Luna C18 column(21.2×250 mm) using a 2%/min gradient from 40-80% MeCN containing 0.1%HCO₂H and 10% H₂O at 20 mL/min. Product containing fractions were pooledand lyophilized to a white microcrystalline powder (224 mg, 0.498 mmol;58.9%). ¹H NMR (DMSO-d₆, 300 MHz): δ 11.15 (1H, s), 8.04 (1H, t, J=6.9),7.29 (4H, ABq, J_(AB)=8.0 Hz), 6.86 (1H, d, J=7.8 Hz), 5.91 (1H, ddt,J=17.4, 10.6, 5.4 Hz), 5.28 (1H, d, J=16.3 Hz), 5.17 (1H, d, J=10.7 Hz),4.73 (2H, s), 4.49 (2H, dt, J=5.4, 1.4 Hz), 4.19 (2H, d, J=6.2), 3.81(1H, AB, J_(AB)=7.9 Hz), 1.60-1.25 (3H, m), 1.37 (9H, s), 0.84 (3H, d,J=6.9 Hz), 0.81 (3H, d, J=6.9 Hz). HRMS calcd for C₂₃H₃₅N₃O₆ (M+Na):472.2418. Found: 472.2415.

Part D—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonylamino]-4-methylpentanamide,formic acid salt

The product of Part 20C (0.200 g, 0.445 mmol) was dissolved in 2:1MeCN/H₂O (8.00 mL) and successively treated with 25.3 mg TPPTS (44.5μmol; 10 mol %), Et₂NH (116 μL, 1.11 mmol), and 5.00 mg Pd(OAc)₂ (22.3μmol; 5 mol %) at 22° C. The resulting yellow solution was stirred 0.5h, then filtered through a 0.45 μm Acrodisk and directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1%/mingradient from 12-37% MeCN containing 0.1% HCO₂H and 10% H₂O at 20mL/min. Product-containing fractions were pooled and lyophilized toafford a white microcrystalline powder (113 mg, 0.275 mmol; 61.7%). ¹HNMR (DMSO-d₆, 600 MHz): δ 8.32 (1H, s), 7.37 (4H, ABq, J_(AB)=8.4 Hz),6.90 (1H, d, J=7.9 Hz), 4.75 (2H, s), 3.85 (2H, s), 3.82 (1H, AB,J_(AB)=8.4 Hz), 1.46-1.56 (1H, m), 1.38 (9H, s), 1.46-1.36 (1H, m),1.36-1.26 (1H, m), 0.85 (3H, d, J=6.5 Hz), 0.82 (3H, d, J=6.2 Hz). LRMS:366.2 (100, M+H), 731.5 (25).

Part E—Preparation of (tert-Butyl2-[(2-{[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-4-methylpentanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl][2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}ethyl){[(tert-butyl)oxycarbonyl]methyl}amino]acetate

A solution of2-{bis[2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}aceticacid (102 mg, 0.166 mmol), HOBt (22.4 mg, 0.166 mmol) and the product ofPart 20D (55.0 mg, 0.150 mmol) in dry DMF (2.00 mL) was successivelytreated with i-Pr₂NEt (115 μL, 0.662 mmol) and HBTU (63.0 mg, 0.166mmol) at 22° C. The resulting solution was stirred 18 h then heated to50° C. and maintained 0.5 h. After cooling to 22° C., all volatiles wereremoved in vacuo and the residue redissolved in EtOAc. The EtOAcsolution was successively washed with 0.1 N HCl, saturated aqueoussolutions of NaHCO₃, and NaCl then dried over Na₂SO₄, filtered andconcentrated in vacuo to a pale yellow oil, which was used withoutfurther purification in the subsequent deprotection step. LRMS: 966.0(100, M+H), 433.6 (60).

Part F—Preparation of2-{[2-({[N-({4-[((2R)-2-amino-4-methylpentanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

The product of Part 20E (0.150 mmol theoretical) was dissolved in 3:2TFA/CH₂Cl₂ (3.00 mL) at 22° C. then stirred overnight. Upon completedeprotection, all volatiles were removed in vacuo and the residuepurified by HPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a2%/min gradient from 0-30% MeCN containing 0.1% TFA and 10% H₂O at 20mL/min. Product-containing fractions were pooled and lyophilized toafford a white microcrystalline powder (85.0 mg, 86.5 μmol; 57.7%). ¹HNMR (DMSO-d₆, 600 MHz) δ 11.76 (1H, s), 8.97 (1H, t, J=5.7 Hz), 8.25(3H, brs), 7.35 (4H, ABq, J_(AB)=8.1 Hz), 4.82 (2H, s), 4.37 (2H, d,J=5.7 Hz), 4.27 (2H, s), 3.50 (9H, brs), 3.38 (4H, t, J=5.6 Hz), 3.06(4H, t, J=5.8 Hz), 1.54-1.47 (3H, m), 0.85 (6H, d, J=5.5 Hz). ¹³C NMR(DMSO-d₆, 151 MHz): δ 172.5, 165.4, 164.6, 138.5, 134.0, 128.8, 127.0,76.7, 54.1, 53.6, 52.0, 48.7, 48.4, 41.8, 23.4, 22.0, 21.8. HRMS calcdfor C₂₈H₄₄N₆O₁₁ (M+H): 641.3141. Found: 641.3450.

Example 212-{[2-({[N-({4-[((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of (R)-Allyl4-(9,9-dimethyl-5-(naphthalen-2-ylmethyl)-4,7-dioxo-2,8-dioxa-3,6-diazadecyl)benzylcarbamate

Prepared as described in Part 20C, using Boc-DNal-OH (126 mg, 0.235mmol; 27.8%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.20 (1H, s), 7.86 (1H, d,J=8.3 Hz), 7.81 (2H, t, J=7.2 Hz), 7.75 (1H, t, J=5.7 Hz), 7.71 (1H, s),7.44-7.50 (2H, m), 7.41 (1H, d, J=8.6 Hz), 7.22 (4H, ABq, J_(AB)=8.1Hz), 7.10 (1H, d, J=8.2 Hz), 5.91 (1H, ddd, J=17.4, 10.7, 5.5 Hz), 5.28(1H, d, J=17.3 Hz), 5.17 (1H, d, J=10.3 Hz), 4.62 (2H, ABq, J_(AB)=11.1Hz), 4.49 (2H, d, J=5.3 Hz), 4.18 (2H, d, J=6.2 Hz), 4.11 (1H, ABq,J_(AB)=8.4 Hz), 2.99 (2H, AB, J_(AB)=7.8 Hz), 1.29 (9H, s). ¹³C NMR(DMSO-d₆, 151 MHz): δ 168.3, 156.1, 155.1, 139.9, 135.4, 133.7, 132.9,131.8, 128.8, 127.7, 127.5, 127.4, 127.3, 126.8, 125.9, 125.4, 116.9,78.0, 76.5, 64.3, 53.5, 43.5, 37.7, 28.1. HRMS calcd forC₃₀H₃₅N₃O₆(M+H): 556.2418. Found: 556.2410.

Part B—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonylamino]-3-(2-naphthyl)propanamide,formic acid salt

Prepared as described in Part 20D (69.0 mg, 0.139 mmol; 60.3%). ¹H NMR(DMSO-d₆, 600 MHz): δ 8.33 (1H, s), 7.86 (1H, d, J=7.8 Hz), 7.82 (1H, t,J=8.3 Hz), 7.72 (1H, s), 7.44-7.50 (2H, m), 7.42 (1H, d, J=8.2 Hz), 7.31(4H, ABq, J_(AB)=7.9 Hz), 7.12 (1H, d, J=7.1 Hz), 4.62 (2H, ABq,J_(AB)=10.8 Hz), 4.12 (1H, m), 3.82 (2H, s), 2.91-3.06 (2H, m), 1.29(9H, s). LRMS: 450.6 (100, M+H).

Part C—Preparation of tert-Butyl2-[(2-{[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-(2-naphthyl)propanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl][2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}ethyl){[(tert-butyl)oxycarbonyl]methyl}amino]acetate

Prepared as described in Part 20E. LRMS: 1050.0 (100, M+H), 618.8 (80),475.6 (45).

Part D—Preparation of2-{[2-({[N-({4-[((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Prepared as described in Part 20F (35.0 mg, 32.8 μmol; 43.2%). ¹H NMR(DMSO-d₆, 300 MHz): δ 11.62 (1H, s), 8.95 (1H, t, J=5.6 Hz), 8.44 (3H,brs), 7.82-7.95 (3H, m), 7.74 (1H, s), 7.56-7.47 (2H, m), 7.37 (1H, d,J=8.8 Hz), 7.17 (4H, ABq J_(AB)=8.1 Hz), 4.55 (2H, AB, J_(AB)=1.0 Hz),4.33 (2H, d, J=5.7 Hz), 4.26 (2H, s), 3.83-3.94 (1H, m), 3.51 (8H, s),3.39 (4H, t, J=5.1 Hz), 3.18 (2H, d, J=7.2 Hz), 3.06 (4H, t, J=4.9 Hz).¹³C NMR (DMSO-d₆, 151 MHz): δ 172.7, 164.8, 164.7, 138.7, 134.1, 132.9,132.3, 132.2, 128.9, 128.2, 128.1, 127.6, 127.5, 127.4, 127.2, 126.3,125.9, 113.8, 77.0, 54.3, 53.9, 52.2, 51.6, 48.6. HRMS calcd forC₃₅H₄₄N₆O₁₁ (M+H): 725.3141. Found: 725.3141.

Example 222-{[2-({[N-({4-[((2R)-2-amino-3-phenylpropanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)-2-[(tert-Butoxy)carbonylamino]-3-phenyl-N-({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methoxy)propanamide

Prepared as described in Part 20C, using Boc-DPhe-OH (88.0 mg, 0.182mmol; 21.5%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.19 (1H, s), 7.76 (1H, t,J=4.2 Hz), 7.32-7.16 (9H, m), 7.02 (1H, d, J=8.3 Hz), 5.91 (1H, ddd,J=17.5, 10.5, 5.4 Hz), 5.28 (1H, d, J=17.1 Hz), 5.17 (2H, d, J=11.5 Hz),4.65 (2H, ABq, J_(AB)=10.7 Hz), 4.49 (2H, d, J=5.3 Hz), 4.19 (2H, d,J=6.2 Hz), 4.00 (1H, ABq, J_(AB)=8.7 Hz), 2.74-2.87 (2H, m), 1.32 (9H,s). HRMS calcd for C₂₆H₃₃N₃O₆ (M+H): 506.2262. Found 506.2254.

Part B—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonylamino]-3-phenylpropanamide,formic acid salt

Prepared as described in Part 20D (45.0 mg, 0.101 mmol; 57.3%). ¹H NMR(DMSO-d₆, 600 MHz): δ 8.33 (1H, s), 7.35 (4H, ABq, J_(AB)=8.3 Hz),7.30-7.17 (5H, m), 7.03 (1H, d, J=8.4 Hz), 4.66 (2H, ABq, J_(AB)=10.6Hz), 3.98-4.04 (1H, m), 3.83 (2H, s), 2.85 (1H, dd, J=13.7, 5.8 Hz),2.78 (1H, dd, J=13.4, 9.5 Hz), 1.32 (9H, s). LCMS: 400.5 (100, M+H).

Part C—Preparation of tert-Butyl2-[(2-{[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-phenylpropanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl][2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}ethyl){[(tert-butyl)oxycarbonyl]methyl}amino]acetate

Prepared as described in Part 20E. LRMS: 1000.0 (100, M+H).

Part D—Preparation of2-{[2-({[N-({4-[((2R)-2-amino-3-phenylpropanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Prepared as described in Part 20F (60.0 mg, 59.0 μmol; 59.7%). ¹H NMR(DMSO-d₆, 300 MHz): δ 11.62 (1H, s), 8.98 (1H, t, J=5.8 Hz), 8.43 (3H,brs), 7.38-7.19 (9H, m), 4.60 (2H, ABq, J_(AB)=10.9 Hz), 4.36 (2H, d,J=5.6 Hz), 4.27 (2H, s), 3.84 (1H, m), 3.51 (8H, s), 3.39 (4H, t, J=5.1Hz), 3.06 (4H, t, J=6.0 Hz), 3.01 (2H, d, J=6.7 Hz). ¹³C NMR (DMSO-d₆,151 MHz): δ 172.7, 164.8, 164.6, 138.7, 134.7, 134.1, 129.4, 129.0,128.6, 127.3, 77.0, 54.3, 53.9, 52.2, 51.5, 48.7, 42.0, 38.6. HRMS calcdfor C₃₁H₄₂N₆O₁₁ (M+Na): 697.2804. Found: 697.2824.

Example 232-(7-{[N-({4-[((2R)-2-amino-4-methylpentanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoracetic acid salt

Part A—Preparation of tert-Butyl2-{7-[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-4-methylpentanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis{[(tert-butyl)oxycarbonyl]methyl}cyclododecyl}acetate

A solution of2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}-cyclododecyl)aceticacid (128 mg, 0.224 mmol) in dry DMF (5.00 mL) was successively treatedwith HOBt (30.3 mg, 0.224 mmol), HBTU (84.9 mg, 0.224 mmol) and i-Pr₂NEt(146 μL, 0.840 mmol) at 22° C. After 0.25 h, the solution was treatedwith the product of Part 20D (55.0 mg, 0.134 mmol) and i-Pr₂NEt (146 μL,0.840 mmol) then stirred overnight. After 24 h, the reaction was heatedto 50° C., maintained 5 h then concentrated in vacuo and the residuedissolved in EtOAc. The EtOAc solution was successively washed with 0.1N HCl, saturated aqueous solutions of NaHCO₃, and NaCl then dried overNa₂SO₄, filtered and concentrated in vacuo to a pale yellow oil, whichwas used without further purification in the subsequent deprotectionstep. LRMS: 921.0 (100, M+H), 411.2 (65).

Part B—Preparation of2-(7-{[N-({4-[((2R)-2-amino-4-methylpentanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoracetic acid salt

The product of Part 23A (0.134 mmol theoretical) was dissolved indioxane (3.00 mL) then successively treated with H₂O (14 μL) and HCl(12.0 mmol; 3.00 mL of a 4 M solution in dioxane) at 22° C. Theresulting pale yellow solution was stirred overnight then all volatilesremoved under reduced pressure and the residue directly purified by HPLCon a Phenomenex Luna C18 column (21.2×250 mm) using a 0.875%/mingradient from 0-35% MeCN containing 0.1% TFA and 10% H₂O at 20 mL/min.Product-containing fractions were pooled and lyophilized to a whitemicrocrystalline powder (63.0 mg, 63.4 μmol; 47.3%). ¹H NMR (DMSO-d₆,600 MHz): 9.01 (1H, t, J=5.4 Hz), 7.42 (4H, ABq, J_(AB)=8.0 Hz), 4.98(2H, s), 4.49 (2H, d, J=5.1 Hz), 3.95 (1H, t, J=6.7 Hz), 3.81 (4H, s),3.80 (2H, s), 3.63 (2H, s), 3.15 (12H, s), 2.99 (4H, s), 1.80-1.66 (3H,m), 0.87 (3H, d, J=6.1 Hz), 0.85 (3H, d, J=6.1 Hz). HRMS calcd forC₃₀H₄₉N₇O₉ (M+H): 652.3665. Found: 652.3669.

Example 242-(7-{[N-({4-[((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

Part A—Preparation of tert-Butyl2-{7-[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-(2-naphthyl)propanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis{[(tert-butyl)oxycarbonyl]methyl}cyclododecyl}acetate

Prepared as described in Part 23A. LRMS: 1005.0 (60, M+H), 453.2 (100).

Part B—Preparation of2-(7-{[N-({4-[((2R)-2-amino-3-(2-naphthyl)propanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

Prepared as described in Part 23B (28.6 mg, 26.5 μmol; 39.9%). ¹H NMR(DMSO-d₆, 600 MHz): δ 8.98 (1H, t, J=5.8 Hz), 7.89-7.79 (5H, m), 7.52(2H, d, J=8.6 Hz), 7.53-7.49 (2H, m), 7.33-7.20 (4H, m), 4.79 (2H, ABq,J_(AB)=11.6 Hz), 4.45 (2H, s), 4.38 (2H, s), 3.80-3.78 (4H, m), 3.59(1H, s), 3.44 (2H, d, J=7.1 Hz), 3.20-3.09 (12H, m), 2.96 (4H, s). HRMScalcd for C₃₇H₄₉N₇O₉ (M+H): 736.3665. Found: 736.3663.

Example 252-{[2-({[N-({4-[((2R)-2-amino-3-indol-2-ylpropanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)-2-[(tert-Butoxy)carbonylamino]-3-indol-2-yl-N-({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methoxy)propanamide

Prepared as described in Part 20C, using Boc-DTrp-OH. LRMS: 423.5 (100,M+H-Boc), 545.5 (15, M+Na).

Part B—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonylamino]-3-indol-2-ylpropanamide,formic acid salt

Prepared as described in Part 20D (154 mg, 0.318 mmol; 43.4%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.22 (1H, brs), 10.80 (1H, s), 8.26 (1H, s), 7.58(1H, d, J=7.7 Hz), 7.36 (1H, d, J=8.0 Hz), 7.31 (4H, ABq, J_(AB)=8.3Hz), 7.12 (1H, s), 7.05 (1H, t, J=7.5 Hz), 6.97 (1H, t, J=7.5 Hz), 6.91(1H, d, J=8.1 Hz), 4.64 (2H, ABq, J_(AB)=11.0 Hz), 4.04 (1H, AB,J_(AB)=7.9 Hz), 3.84 (2H, s), 2.99 (1H, dd, J=14.3, 5.9 Hz), 2.90 (1H,dd, J=14.4, 8.8 Hz), 1.33 (9H, s). HRMS calcd for C₂₄H₃₀N₄O₄ (M+Na):461.259. Found: 461.259.

Part C—Preparation of tert-Butyl2-[(2-{[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-indol-2-ylpropanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl][2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}ethyl){[(tert-butyl)oxycarbonyl]methyl}amino]acetate

Prepared as described in Part 20E to afford the crude product which wasused without purification in the next step. LRMS (m/z): 1039.0 (100%,[M+H]⁺)

Part D—Preparation of2-{[2-({[N-({4-[((2R)-2-amino-3-indol-2-ylpropanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Prepared as described in Part 20F (47.7 mg, 45.2 μmol; 26.1%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.63 (1H, s), 11.02 (1H, s), 8.95 (1H, t, J=5.6Hz), 8.28 (2H, brs), 7.60 (1H, d, J=7.9 Hz), 7.38 (1H, d, J=8.1 Hz),7.25 (4H, ABq, J_(AB)=8.1 Hz), 7.12 (1H, s), 7.10 (1H, t, J=7.4 Hz),6.91 (1H, t, J=7.5 Hz), 4.60 (2H, ABq, J_(AB)=10.9 Hz), 4.36 (2H, d,J=4.7 Hz), 4.26 (2H, s), 3.78-3.72 (1H, m), 3.51 (8H, s), 3.38 (4H, t,J=5.3 Hz), 3.18 (1H, dd, J=14.4, 7.2 Hz), 3.10 (1H, dd, J=14.4, 7.4 Hz),3.01 (4H, t, J=5.4 Hz). HRMS calcd for C₃₃H₄₃N₇O₁₁ (M+H): 714.3093.Found: 714.3089.

Example 262-(4-{[N-({4-[((2R)-2-amino-3-indol-2-ylpropanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-7,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

Part A—Preparation of tert-Butyl2-{10-[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-indol-2-ylpropanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,7-bis{[(tert-butyl)oxycarbonyl]methyl}cyclododecyl}acetate

Prepared as described in Part 23A. LRMS: 994.0 (100, M+H), 589.7 (50),447.6 (100).

Part B—Preparation of(2-(4-{[N-({4-[((2R)-2-amino-3-indol-2-ylpropanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}-1,4,7,10-tetraaza-7,10-bis(carboxymethyl)cyclododecyl)aceticacid, trifluoroacetic acid salt

Prepared as described in Part 23B (13 mg, 11 μmol; 8.4%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.63 (1H, s), 11.03 (1H, s), 8.95 (1H, brs), 8.31(2H, brs), 7.60 (1H, d, J=7.9 Hz), 7.38 (1H, d, J=8.1 Hz), 7.25 (4H,ABq, J_(AB)=7.9 Hz), 7.20 (1H, s), 7.10 (1H, t, J=7.5 Hz), 7.02 (1H, t,J=7.5 Hz), 4.60 (2H, ABq, J_(AB)=10.9 Hz), 4.36 (2H, d, J=4.5 Hz), 3.75(1H, t, J=6.4 Hz), 3.63 (4H, s), 3.35 (12H, brs), 3.17 (1H, dd, J=14.4,7.2 Hz), 3.10 (1H, dd, J=14.5, 7.5 Hz), 3.04 (8H, brs). HRMS calcd forC₃₅H₄₈N₈O₉ (M+H): 725.3617. Found: 725.3627.

Example 272-({2-[({N-[(4-{[(2R)-2-amino-3-(4-hydroxyphenyl)propanoylaminooxy]methyl}phenyl)methyl]carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)amino)aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)-2-[(tert-Butoxy)carbonylamino]-3-(4-hydroxyphenyl)-N-({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methoxy)propanamide

Prepared as described in Part 20C, using Boc-DTyr-OH (33 mg, 66 μmol;7.8%). ¹H NMR (DMSO-d₆, 600 MHz): δ 11.14 (1H, s), 9.14 (1H, s), 7.76(1H, t, J=6.0 Hz), 7.27 (4H, ABq, J_(AB)=7.9 Hz), 7.00 (2H, d, J=8.2Hz), 6.93 (1H, d, J=8.5 Hz), 6.64 (2H, d, J=8.3 Hz), 5.91 (1H, ddt,J=17.5, 10.4, 5.1 Hz), 5.28 (1H, dd, J=17.2, 1.3 Hz), 5.17 (1H, dd,J=10.7, 1.0 Hz), 4.64 (2H, ABq, J_(AB)=10.9 Hz), 4.49 (2H, dt, J=5.5,1.3 Hz), 4.19 (2H, d, J=6.2), 3.91 (1H, AB, J_(AB)=8.4 Hz), 2.62-2.76(2H, m), 1.33 (9H, s). HRMS calcd for C₂₆H₃₃N₃O₇(M+H): 522.2211. Found:522.2203.

Part B—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonylamino]-3-(4-hydroxyphenyl)propanamide,formic acid salt

Prepared as described in Part 20D (16.9 mg, 36.6 μmol; 59.0%). ¹H NMR(DMSO-d₆, 600 MHz): δ 8.36 (1H, brs), 7.52 (1H, dt, J=7.6, 2.8 Hz), 7.40(4H, ABq, J_(AB)=7.7 Hz), 7.00 (2H, d, J=8.1 Hz), 6.93 (1H, d, J=8.3Hz), 6.64 (2H, d, J=7.9 Hz), 4.68 (2H, ABq, J_(AB)=10.9 Hz), 3.97 (2H,s), 3.91 (1H, AB, J_(AB)=8.0 Hz), 2.73 (1H, dd, J=13.7, 5.9 Hz), 2.66(1H, dd, J=13.3, 9.2 Hz), 1.33 (9H, s). HRMS calcd for C₂₂H₂₉N₃O₅(M+H):416.2180. Found: 416.2183.

Part C—Preparation of tert-Butyl2-[(2-{[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-(4-hydroxyphenyl)propanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl][2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}ethyl){[(tert-butyl)oxycarbonyl]methyl}amino]acetate

Prepared as described in Part 20E. LRMS: 1016.0 (45, M+H), 458.6 (30,(M−Boc)+2H).

Part D—Preparation of2-({2-[({N-[(4-{[(2R)-2-amino-3-(4-hydroxyphenyl)propanoylaminooxy]methyl}phenyl)methyl]carbamoyl}methyl){2-[bis(carboxymethyl)amino]ethyl}amino]ethyl}(carboxymethyl)amino)aceticacid, trifluoroacetic acid salt

Prepared as described in Part 20F. ¹H NMR (DMSO-d₆, 600 MHz): δ 11.54(1H, s), 9.37 (1H, brs), 8.95 (1H, t, J=5.3 Hz), 8.32, (1H, brs), 8.28(1H, brs), 7.30 (4H, ABq, J_(AB)=8.2 Hz), 7.00 (2H, d, J=8.2 Hz), 6.72(2H, d, J=8.6 Hz), 4.64 (2H, ABq, J_(AB)=10.9 Hz), 4.36 (2H, d, J=5.9Hz), 4.26 (2H, s), 3.66 (2H, brs), 3.66-3.39 (9H, m), 3.41-3.36 (4H, m),3.06 (4H, t, J=5.6 Hz), 2.88 (2H, d, J=7.6 Hz). HRMS calcd forC₃₁H₄₂N₆O₁₂ (M+H): 691.2936. Found: 691.2944.

Example 282-{[2-({[N-({4-[((2R)-2-amino-3-(3-pyridyl)propanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Part A—Preparation of(2R)-2-[(tert-butoxy)carbonylamino]-N-({4-[(prop-2-enyloxycarbonylamino)methyl]phenyl}methoxy)-3-(3-pyridyl)propanamide

Prepared as described in Part 20C, using Boc-DPya-OH. LRMS: 485.6 (100,M+H).

Part B—Preparation of(2R)—N-{[4-(aminomethyl)phenyl]methoxy}-2-[(tert-butoxy)carbonylamino]-3-(3-pyridyl)propanamide,formic acid salt

Prepared as described in Part 20D (151 mg, 0.338 mmol; 79.9%). LRMS:401.6 (100, M+H).

Part C—Preparation of tert-Butyl2-[(2-{[(N-{[4-({(2R)-2-[(tert-butoxy)carbonylamino]-3-(3-pyridyl)propanoylaminooxy}methyl)phenyl]methyl}carbamoyl)methyl][2-(bis{[(tert-butyl)oxycarbonyl]methyl}amino)ethyl]amino}ethyl){[(tert-butyl)oxycarbonyl]methyl}amino]acetate

Prepared as described in Part 20E. LRMS: 1001.0 (75, M+H), 501.2 (100,M+2H).

Part D—Preparation of2-{[2-({[N-({4-[((2R)-2-amino-3-(3-pyridyl)propanoylaminooxy)methyl]phenyl}methyl)carbamoyl]methyl}{2-[bis(carboxymethyl)amino]ethyl}amino)ethyl](carboxymethyl)amino}aceticacid, trifluoroacetic acid salt

Prepared as described in Part 20F (3.3 mg, 3.2 μmol; 1.8%). ¹H NMR(DMSO-d₆, 600 MHz): δ 11.64 (1H, s), 8.95 (1H, t, J=5.8 Hz), 8.52 (1H,d, J=5.0 Hz), 8.38 (2H, brs), 7.81-7.76 (1H, m), 7.38-7.26 (6H, m), 4.69(2H, ABq, J_(AB)=11.6 Hz), 4.37 (2H, d, J=4.7 Hz), 4.27 (2H, s), 4.07(1H, t, J=5.9 Hz), 3.64 (8H, brs), 3.39 (4H, t, J=5.7 Hz), 3.19 (2H, d,J=7.1 Hz), 3.06 (4H, t, J=5.9 Hz). HRMS calcd for C₃₀H₄₁N₇O₁₁ (M+H):676.2937. Found: 676.2940.

Examples 29-36 Synthesis of Gadolinium Complexes

The following procedure is representative of the fashion in whichgadolinium complexes of the aforementioned examples are prepared. Yieldand characterization data are provided in Table 1.

A solution of the product of Example 2 (24.3 mg, 23.3 μmol) in Milli-QH₂O (466 μL) was treated with GdCl₃ (7.4 mg, 28 μmol) in one portion at22° C. The pH of the solution was adjusted to 5-6 with aqueous NaOH (933μL of a 0.1 M solution); direct HPLC analysis of the reaction mixtureusing a pH 7 mobile phase indicated complexation was complete. Thesolution was diluted with 15 mM NH₄OAc (5 mL) and directly purified byHPLC on a Phenomenex Luna C18 column (21.2×250 mm) using a 1.0%/mingradient of 0-30% MeCN at a flow rate of 20 mL/min; 5 mM NH₄OAc wasemployed as the aqueous component. The main product peak eluting at 19min was collected and lyophilized to give the title compound as amicrocrystalline solid (15.5 mg, 18.2 μmol; 77.8%).

TABLE 1 Characterization data for Examples 29-36 precursor (as shown inHRMS example Example #) yield (%) LRMS (ESI) (calcd. for; found) 29 1 511687.1 (21, 2M + H), 1265.3 (20, C₃₂H₄₁GdN₆O₁₁ (M + H) 3M + 2H), 843.8(100, M + H) 844.2147; 844.2140 30 2 78 855.6 (100, M + H), 428.5 (24,M + 2H) C₃₄H₄₆GdN₇O₉ (M + H) 855.2671; 855.2681 31 23 64 1614.1 (12,2M + H), 807.6 (100, M + H), C₃₀H₄₆GdN₇O₉ (M + H) 403.5 (45, M + 2H)807.2671; 807.2678 32 21 43 1320 (27, 3M + 2H), 880.3 (100, M + H),C₃₅H₄₁GdN₆O₁₁ (M + H) 441.7 (72, M + 2H) 880.2147; 880.2155 33 24 16891.6 (79, M + H), 736.8 (100), 369.0 (54) C₃₇H₄₆GdN₇O₉ (M + H)891.2671; 891.2677 34 20 69 1591.9 (15, 2M + H), 796.5 (100, M + H),C₂₈H₄₁GdN₆O₁₁ (M + H) 398.9 (55, M + 2H) 796.2147; 796.2148 35 25 55880.7 (21, M + Na), 869.1 (100, M + H), C₃₃H₄₀GdN₇O₁₁ (M + H) 435.8 (25,M + 2H) 869.2100; 869.2099 36 26 15 1759.2 (10, 2M + H), 880.7 (100, M +H), C₃₅H₄₅GdN₈O₉ (M + H) 440.0 (35, M + 2H) 880.2623; 880.2625

Examples 37-64 Synthesis of [¹⁵³Gd]Gadolinium Complexes

The following procedure is representative of the fashion in whichgadolinium complexes of the aforementioned examples are prepared.Radiochemical purity values for each complex are provided in Table 2.

Using a lead-shielded vial, a solution of the product of Example 2(0.350 mg, 0.336 μmol) in 0.5 M NH₄OAc (0.850 mL) was treated with[¹⁵³Gd]GdCl₃ (75 μL of a 12.5 mCi/μL solution in 0.5 N HCl) in oneportion at 22° C. The vial was capped using a rubber stopper, securedwith an aluminum crimp ring, then heated to 95° C. (H₂O bath) andmaintained 20 min. After cooling to 22° C., a 25 μL aliquot was removedand analyzed by HPLC to confirm complete conversion. The crude reactionmixture was then purified by HPLC on a Phenomenex Cosmosil C18 column(4.6×250 mm) using a 6.7%/min gradient from 0-100% MeCN at 1 mL/min withdetection using inline INUS β-Ram and PDA (220 nm) modules; 25 mM NH₄OAcwas employed as the aqueous component. Product-containing fractions werecollected, concentrated under reduced pressure and analyzed using theaforementioned method to determine radiochemical purity.

TABLE 2 Characterization data for Examples 37-64 precursor (as shown inExample #) Example # % RCP 1 37 100 2 38 100 3 39 100 4 40 100 5 41 95.06 42 98.7 7 43 99.1 8 44 96.6 9 45 99.3 10 46 74.5 11 47 96.3 12 48 68.213 49 100 14 50 100 15 51 100 16 52 95.4 17 53 80.0 18 54 100 19 55 10020 56 100 21 57 96.9 22 58 98.8 23 59 94.5 24 60 100 25 61 100 26 62 10027 63 100 28 64 100

Example 65 Ex-Vivo Blood Vessel Binding Assay

Aorta bearing atherosclerotic plaque was obtained from New Zealand whiterabbits that were balloon stripped along the abdominal aorta and placedon a high fat diet (0.5% cholesterol) for 16-22 weeks. Vascular injurywas produced with a 4-F Fogarty catheter along the abdominal aorta andright iliofemoral artery. This procedure generates an acceleratedcomplex lesion development with a lipid rich core covered by a fibrouscap in rabbits. Harvested aorta sections (0.5 cm) were incubated with0.135 μCi of ¹⁵³Gd-labeled compound diluted in phosphate buffered saline(450 μL) for 2 h at 37° C. The supernatant was removed and analyzed byHPLC to assay compound stability. The tissue section was then washedwith phosphate buffered saline (3×10 mL), then resuspended (10 mL) andincubated at 37° C. an additional 1 h. The supernatant was then removed,the washing process repeated and the tissue finally counted on a gammacounter. The amount of compound bound to the tissue was determined as apercentage of the initial activity according to the following formula:

${\%\mspace{14mu}{Tissue}\mspace{14mu}{Uptake}} = {\frac{{Counts}\mspace{14mu}{bound}\mspace{14mu}{to}\mspace{14mu}{tissue}}{{Total}\mspace{14mu}{counts}\mspace{14mu}{in}\mspace{14mu}{test}\mspace{14mu}{tube}} \times 100}$

The data for percentage compound bound to plaque-bearing aorta iscollected in Table 3.

TABLE 3 Ex-vivo blood vessel binding data example # % bound 37 12.8 3819.1 39 15.8 40 7.2 41 28.7 42 17.9 43 27.1 44 27.3 45 14.4 46 0.9 476.6 49 9.1 50 24.2 52 6.9 57 30.6 58 7.3 59 5.2 60 30.9 61 19.9 62 17.163 11.7

Example 66 In-Vivo ApoE Mouse Aorta Uptake Studies

The apolipoprotein E (ApoE) knockout mouse is a model ofhypercholesterolemia that develops atherosclerotic lesions in thebrachiocephalic artery, the aortic arch and the abdominal aorta. Micewere fed a high-fat diet to accelerate plaque formation and compoundswere tested in the mice between 35-42 weeks on diet. Test compounds wereadministered at 0.3-0.4 mCi/kg to anesthetized mice in a single, bolusinjection via the tail vein. Blood samples were collected via the tailbetween 0-30 min post injection for pharmacokinetic analysis and micewere euthanized by CO₂ at 60 min for tissue harvesting. The aorta wasfirst flushed with saline through the left ventricle exiting via thefemoral vein then removed from the heart to the renal bifurcation;additional biological samples were also collected (blood, muscle, liver,kidney, bile, urine, heart, femur, reproductive organ, lung, spleen andinnominate artery). All samples were weighed and assayed forradioactivity; uptake is expressed as a percentage of injected dose pergram of tissue (% ID/g). Aorta uptake, aorta to blood ratios and aortato heart ratios are summarized in Table 4.

TABLE 4 ApoE mouse aorta uptake, aorta:heart and aorta:blood ratiosaorta uptake example # (% ID/g) aorta: heart aorta: blood 37 10.4 ± 1.4 16.2 4.2 38 11.6 ± 0.8  20.5 5.5 39 6.1 ± 0.4 13.6 11.4 40 3.6 ± 0.3 8.43.4 41 2.0 ± 0.1 20.4 7.2 42 8.1 ± 1.1 18.0 8.0 43 6.0 ± 1.7 9.0 2.7 448.4 ± 0.2 12.5 3.2 45 9.0 ± 0.2 12.1 2.1 46 0.9 ± 0.2 4.9 1.1 47 4.7 ±0.4 4.8 0.7 49 4.9 ± 0.2 22.9 5.5 50 10.4 ± 2.4  13.5 3.7 52 6.1 ± 1.29.2 2.0 53 1.5 ± 0.1 4.2 0.8 54 2.5 ± 0.5 5.3 1.2 55 3.0 ± 2.3 8.8 1.056 8.5 ± 1.8 21.2 5.5 57 11.1 ± 0.3  17.8 5.0 58 6.1 ± 0.2 17.9 6.5 598.2 ± 1.3 15.4 3.7 60 18.4 ± 2.4  25.2 9.8 61 19.3 ± 0.2  20.4 10.9 6211.8 ± 1.4  22.2 5.7 63 8.7 ± 0.4 13.0 6.0

Example 67 In-Vivo Rabbit Aorta Uptake Studies

Atherosclerosis was induced in New Zealand White male rabbits (3 kg)with aortic balloon endothelial injury (vide supra) followed by feedinga 0.5% cholesterol diet for 22 weeks. Test compounds were administeredat 0.01-0.05 mCi/kg to anesthetized rabbits in a single, bolus injectionvia the marginal ear vein. Blood samples were collected from the centralear artery at 0, 2, 5, 7, 10, 15, 30 and 60 min post injection. Rabbitswere euthanized at 60 min post injection for tissue harvesting (blood,muscle, bile, urine, kidney, liver, spleen, heart, lung, colon, smallintestine, stomach, testes and in some cases, sternum, ligament andright ear). Abdominal aorta (upper, middle, and lower) and left andright femoral arteries were also collected. All samples were weighed andassayed for radioactivity; uptake is expressed as percentage of injecteddose per gram of tissue (% ID/g). Aorta uptake, aorta to blood ratiosand aorta to heart ratios are summarized in Table 5; a comparativeanalysis between plaque bearing and non-plaque bearing rabbits is alsoprovided.

TABLE 5 Rabbit aorta uptake, aorta:heart and aorta:blood ratios plaquerabbit control rabbit aorta aorta example # (% ID/g ± SD) aorta:heartaorta:blood (% ID/g ± SD) aorta:heart aorta:blood 37 0.087 ± 0.010 4.62.1 — — — 38 0.103 ± 0.002 5.1 2.4 0.196 ± 0.023 11.0  5.1 60 0.108 ±0.012 4.0 2.4 0.187 ± 0.022 6.4 3.8 62 — — — 0.159 6.4 3.2

Example 68 In Vivo Rabbit Aorta MR Imaging

Atherosclerosis was induced in New Zealand White male rabbits (3 kg)with aortic balloon endothelial injury (vide supra) followed by feedinga 0.5% cholesterol diet for 22 weeks. A series of pre-injection imageswere acquired. The rabbit was then injected with test compound (i.e.,Example 31) at 0.1 mmol/kg via the marginal ear vein and images acquiredat specified time intervals. All images were acquired at 4.7 T using an8.5 cm field of view, 256×256 matrix using a black blood,flow-suppressed spin-echo method. A marked increase of relative imageintensity in the aorta (ring-shaped structure) was observed shortlyafter injection; sample images are provided in FIG. 1.

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. A diagnostic agent, comprising: a compound of Formula (I),

or a pharmaceutically acceptable salt thereof, wherein: X is N, O, S, orP; R¹ is hydrogen, alkyl, alkenyl, arylalkyl, alkylarylalkyl,alkoxyalkyl, heteroalkyl, or heterocyclylalkyl, provided that when X isN, at least one R¹ is H; R² and R³ can be the same or different and arehydrogen, alkyl, alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl,arylalkyl, alkylarylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonyl,heteroalkyl, heterocyclyl, heterocyclylalkyl, or carbonyl; and R⁴ isalkyl, alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl,alkylarylalkyl, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,or heterocyclylalkyl; wherein each R¹, R², R³, and R⁴ is unsubstitutedor substituted with one or more of the following: alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl, orheterocyclylalkyl, —NR¹⁹R²⁰, —SH, —S(Pg), —OH, —PR¹⁹R²⁰, —P(O)R²¹R²²,—CO₂H, ═O, halo, trifluoromethyl, cyano, —CO₂R²⁴, —C(═O)R²⁴,—C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴, —OR²⁴,—OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂,—NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴, —S(═O)R²⁴,—SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂, —C(═O)NHOR²⁴,—C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy, or a chelatormoiety; R¹⁹ and R²⁰ are each independently selected from hydrogen,C₁₋₁₀alkyl substituted with 0-3 R²³, aryl substituted with 0-3 R²³,C₃-C₁₀cycloalkyl substituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkylsubstituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³,and heterocyclyl substituted with 0-3 R²³; R²¹ and R²² are eachindependently selected from —OH, C₁₋₁₀alkyl substituted with 0-3 R²³,aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substituted with 0-3 R²³,heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl; eachR²⁴ is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; Pg is a thiol protecting group; and n′is an integer from 1-3, wherein the compound of Formula (I) comprises atleast one chelator moiety; and an imaging agent bound to the at leastone chelator moiety, and wherein at least one of R¹, R², R³, or R⁴ issubstituted with the at least one chelator moiety.
 2. The diagnosticagent of claim 1, wherein the imaging agent is an echogenic substance,an optical reporter, a boron neutron absorber, a paramagnetic metal ion,a ferromagnetic metal, a gamma-emitting radioisotope, apositron-emitting radioisotope, or an x-ray absorber.
 3. The diagnosticagent of claim 2, wherein the imaging agent is a paramagnetic metal ion.4. The diagnostic agent of claim 3, wherein the paramagnetic metal ionis optionally Gd(III).
 5. The diagnostic agent of claim 2, wherein theimaging agent is a gamma-emitting radioisotope or positron-emittingradioisotope selected from the group consisting of ¹¹¹In, ⁶²Cu, ⁶⁴Cu,⁶⁷Ga, ⁶⁸Ga, and ¹⁵³Gd.
 6. The diagnostic agent of claim 2, wherein thediagnostic agent has a structure selected from the group consisting of:


7. A method of detecting, imaging, and/or monitoring a patientcomprising the steps of: administering to the patient a diagnostic agentof claim 1; and acquiring an image of a site of concentration of thediagnostic agent in the patient by a diagnostic imaging technique. 8.The method of claim 7, wherein the method comprises detecting, imaging,and/or monitoring elastin-rich tissues in a patient.
 9. The method ofclaim 7, wherein the method comprises detecting, imaging, and/ormonitoring the presence of coronary plaque, carotid plaque,iliac/femoral plaque, aortic plaque, renal artery plaque, plaque of anyarterial vessel, aneurism, vasculitis, other diseases of the arterialwall, and/or damage or structural changes in ligaments, uterus, lungs orskin in a patient.
 10. The diagnostic agent of claim 1, wherein X isoxygen.
 11. The diagnostic agent of claim 1, wherein X is nitrogen. 12.The diagnostic agent of claim 1, wherein: X is nitrogen; R¹ is hydrogen,alkyl, arylalkyl, or alkylarylalkyl; R² and R³ can be the same ordifferent and are hydrogen, alkyl, alkylaryl, aryl, arylalkyl,alkylarylalkyl, or heterocyclylalkyl; R⁴ is alkyl, alkylaryl, aryl,arylalkyl, or alkylarylalkyl, wherein each R¹, R², R³, and R⁴ isunsubstituted or substituted with one or more of the following: alkyl,alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl,alkylarylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl,heterocyclyl, or heterocyclylalkyl, —NR¹⁹R²⁰, —SH, —S(Pg), —OH,—PR¹⁹R²⁰, —P(O)R²¹R²², —CO₂H, ═O, halo, trifluoromethyl, cyano, —CO₂R²⁴,—C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴,—OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂,—NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴, —S(═O)R²⁴,—SO₂N(R²⁴)₂, —N(R²⁴)₂, NHC(═S)NHR²⁴, ═NOR²⁴, NO₂, —C(═O)NHOR²⁴,—C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy, or a chelatormoiety; R¹⁹ and R²⁰ are each independently selected from hydrogen,C₁₋₁₀alkyl substituted with 0-3 R²³, aryl substituted with 0-3 R²³,C₃₋₁₀cycloalkyl substituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkylsubstituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³,and heterocyclyl substituted with 0-3 R²³; R²¹ and R²² are eachindependently selected from —OH, C₁₋₁₀alkyl substituted with 0-3 R²³,aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substituted with 0-3 R²³,heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl; eachR²⁴ is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; Pg is a thiol protecting group; andwherein at least one of R¹, R², R³, and R⁴ is substituted with achelator moiety.
 13. The diagnostic agent of claim 1, wherein: R² or R³comprises the following structure,

wherein: n is 0-6; and R^(z) is alkyl, aryl, cycloalkyl, orheterocyclyl.
 14. The diagnostic agent of claim 1, wherein:

comprises the following structure,

wherein: n is 0-6; R^(y) is hydrogen, alkenyl, or alkyl; and R^(z) isalkyl, aryl, cycloalkyl, or heterocyclyl.
 15. The diagnostic agent ofclaim 1, wherein:

comprises the following structure,

wherein: n is 1 or 2; R^(y) is hydrogen; and R^(z) is alkyl or aryl. 16.The diagnostic agent of claim 1, wherein R¹ is substituted with the atleast one chelator moiety.
 17. The diagnostic agent of claim 1, whereinR² or R³ is substituted with the at least one chelator moiety.
 18. Thediagnostic agent of claim 1, wherein R⁴ is substituted with the at leastone chelator moiety.
 19. The diagnostic agent of claim 1, wherein thecompound of Formula (I) has a structure as in Formula (II),

or a pharmaceutically acceptable salt thereof, wherein: R⁴ is alkyl,alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl,alkylarylalkyl, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,or heterocyclylalkyl, substituted with the at least one chelator moiety;n is 0-6; R^(y) is hydrogen, alkenyl, or alkyl; and R^(z) is alkyl,aryl, cycloalkyl, or heterocyclyl, wherein R⁴ is unsubstituted orsubstituted with one or more of the following: alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl, orheterocyclylalkyl, —NR¹⁹R²⁰, —SH, —S(Pg), —OH, —PR¹⁹R²⁰, —P(O)R²¹R²²,—CO₂H, ═O, halo, trifluoromethyl, cyano, —CO₂R²⁴, —C(═O)R²⁴,—C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴, —OR²⁴,—OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂,—NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴, —S(═O)R²⁴,—SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂, —C(═O)NHOR²⁴,—C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy, or a chelatormoiety; R¹⁹ and R²⁰ are each independently selected from hydrogen,C₁₋₁₀alkyl substituted with 0-3 R²³, aryl substituted with 0-3 R²³,C₃₋₁₀cycloalkyl substituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkylsubstituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³,and heterocyclyl substituted with 0-3 R²³; R²¹ and R²² are eachindependently selected from —OH, C₁₋₁₀alkyl substituted with 0-3 R²³,aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substituted with 0-3 R²³,heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl; eachR²⁴ is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; and Pg is a thiol protecting group. 20.The diagnostic agent of claim 1, wherein the compound of Formula (I) hasa structure as in Formula (III),

or a pharmaceutically acceptable salt thereof, wherein: R² and R³ can bethe same or different and are hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl, and at least one of R² and R³ issubstituted with the at least one chelator moiety; and R⁴ is alkyl,alkylaryl, or arylalkyl, wherein each R², R³, and R⁴ is unsubstituted orsubstituted with one or more of the following: alkyl, alkenyl,cycloalkyl, alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl, orheterocyclylalkyl, —NR¹⁹R²⁰, —SH, —S(Pg), —OH, —PR¹⁹R²⁰, —P(O)R²¹R²²,—CO₂H, ═O, halo, trifluoromethyl, cyano, —CO₂R²⁴, —C(═O)R²⁴,—C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR²⁴, —OR²⁴,—OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴, —NR²⁴C(═O)N(R²⁴)₂,—NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴, —S(═O)R²⁴,—SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂, —C(═O)NHOR²⁴,—C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy, or a chelatormoiety; R¹⁹ and R²⁰ are each independently selected from hydrogen,C₁₋₁₀alkyl substituted with 0-3 R²³, aryl substituted with 0-3 R²³,C₃₋₁₀cycloalkyl substituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkylsubstituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³,and heterocyclyl substituted with 0-3 R²³; R²¹ and R²² are eachindependently selected from —OH, C₁₋₁₀alkyl substituted with 0-3 R²³,aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substituted with 0-3 R²³,heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl; eachR²⁴ is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; and Pg is a thiol protecting group. 21.The diagnostic agent of claim 1, wherein the compound of Formula (I) hasa structure as in Formula (IV),

or a pharmaceutically acceptable salt thereof, wherein: R¹ is alkyl,alkenyl, cycloalkyl, arylalkyl, alkylarylalkyl, alkoxyalkyl,heteroalkyl, or heterocyclylalkyl, substituted with the at least onechelator moiety; n is 0-6; R^(z) is alkyl, aryl, cycloalkyl, orheterocyclyl; and R⁴ is alkyl, alkylaryl, or arylalkyl, wherein each R¹and R⁴ is unsubstituted or substituted with one or more of thefollowing: alkyl, alkenyl, cycloalkyl, alkylaryl, alkylcarbonyl, aryl,arylalkyl, alkylarylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonyl,heteroalkyl, heterocyclyl, or heterocyclylalkyl, —NR¹⁹R²⁰, —SH, —S(Pg),—OH, —PR¹⁹R²⁰, P(O)R²¹R²², —CO₂H, ═O, halo, trifluoromethyl, cyano,—CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂,—C(═O)NHOR²⁴, —C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy, or achelator moiety; R¹⁹ and R²⁰ are each independently selected fromhydrogen, C₁₋₁₀alkyl substituted with 0-3 R²³, aryl substituted with 0-3R²³, C₃₋₁₀cycloalkyl substituted with 0-3 R²³, heterocyclyl-C₁₋₁₀alkylsubstituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkyl substituted with 0-3 R²³,and heterocyclyl substituted with 0-3 R²³; R²¹ and R²² are eachindependently selected from —OH, C₁₋₁₀alkyl substituted with 0-3 R²³,aryl substituted with 0-3 R²³, C₃₋₁₀cycloalkyl substituted with 0-3 R²³,heterocyclyl-C₁₋₁₀alkyl substituted with 0-3 R²³, C₆₋₁₀aryl-C₁₋₁₀alkylsubstituted with 0-3 R²³, and heterocyclyl substituted with 0-3 R²³;each R²³ is independently selected from ═O, halo, trifluoromethyl,cyano, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO, —CH₂OR²⁴, —OC(═O)R²⁴,—OC(═O)OR²⁴, —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁴C(═O)R²⁴, —NR²⁴C(═O)OR²⁴,—NR²⁴C(═O)N(R²⁴)₂, —NR²⁴SO₂N(R²⁴)₂, —NR²⁴SO₂R²⁴, —SO₃H, —SO₂R²⁴, —SR²⁴,—S(═O)R²⁴, —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, —NO₂,—C(═O)NHOR²⁴, —C(═O)NHN(R²⁴)₂, —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁₋₅alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl, C₃₋₆cycloalkylmethyl,C₂₋₆alkoxyalkyl, aryl substituted with 0-2 R²⁴, and heterocyclyl; eachR²⁴ is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,alkylaryl, alkylcarbonyl, aryl, arylalkyl, alkylarylalkyl, alkoxy,alkoxyalkyl, alkoxycarbonyl, heteroalkyl, heterocyclyl,heterocyclylalkyl, or carbonyl; and Pg is a thiol protecting group. 22.The diagnostic agent as in claim 1, wherein the at least one chelatormoiety has the structure,

wherein X′ is a heteroatom and wherein one of D¹ and D² is a hydrogenand the other is a chelator moiety.
 23. The diagnostic agent of claim22, wherein X′ is nitrogen.
 24. The diagnostic agent of claim 22,wherein one of D¹ and D² is hydrogen and the other is selected from thegroup consisting of:

wherein o, p, q, r, s, t, and u are each independently 1-6; and v, w, x,and y are each independently 1-3; or wherein o, r, s, t, and u are each1; and p and q are each 2; or wherein o, r, s, t, v, w, x and y areeach
 1. 25. The diagnostic agent of claim 1, wherein at least one R¹ ishydrogen.
 26. The diagnostic agent of claim 1, wherein X is sulfur. 27.The diagnostic agent of claim 1, wherein X is phosphorus.